Glucagon antagonist-gip agonist conjugates and compositions for the treatment of metabolic disorders and obesity

ABSTRACT

Provided herein are peptide combinations comprising a GIP agonist peptide and a glucagon antagonist peptide. In some embodiments, the peptide combination is provided as a composition, e.g., a pharmaceutical composition, while in other embodiments, the peptide combination is provided as a kit. In yet other embodiments, the peptide combination is provided as a conjugate, e.g., a fusion peptide, a heterodimer. In specific aspects, the GIP agonist peptide is an analog of native human glucagon. In specific aspects, the glucagon antagonist peptide is an analog of native human glucagon. In some embodiments, the GIP agonist peptide is covalently attached to the glucagon antagonist peptide via a linker. Methods of treating a disease, e.g., a metabolic disorder, such as diabetes and obesity, comprising administering the peptide compositions described herein are further provided.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing (organized into Sequence Listings1-5, as described herein) submitted concurrently herewith and identifiedas follows: One 723,000 byte ACII (Text) file named“31135-45097_ST25.txt,” created on Jan. 27, 2010.

BACKGROUND

Glucose-dependent insulinotropic peptide (GIP) and Glucagon-likePeptide-1 (GLP-1) are incretins which regulate the amount of insulinthat is secreted after eating (Kim and Egan, Pharm Rev 60:470-512(2008)). In specific, GIP exerts glucose-dependent stimulatory effectson insulin secretion, thereby ensuring prompt insulin-mediated uptake ofglucose into tissues, and GLP-1 stimulates insulin synthesis andsecretion, inhibition of glucagon secretion, and inhibition of foodintake. While agonist peptide analogs of both incretins have been madeand tested, GLP-1 agonists have been and remain the central focus ofresearch and development for treating of metabolic diseases, such asType 2 diabetes. This is not surprising, since in Type 2 diabetes, GIPno longer modulates glucose-dependent insulin secretion, whereas GLP-1retains insulinotropic activities even in Type 2 diabetic patients.Also, research by some groups McLean et al., Am J Physiol EndocrinolMetab 296(6): E1746-1755 (epub 2007) have suggested the use of GIPantagonists and not GIP agonists for the treatment of diabetes.

When blood glucose levels begin to fall, glucagon is produced by thepancreas and the binding of this hormone to its receptor signals theliver to break down glycogen and release glucose. The actions ofglucagon cause blood glucose levels to rise toward a normal level.Because glucagon exerts actions which oppose incretins, many glucagonantagonists have been made and tested for the treatment of metabolicdiseases, including Type 2 diabetes.

SUMMARY

Provided herein are peptide combinations useful for the treatment ofdiseases, such as metabolic disorders (e.g., diabetes, obesity). Thepeptide combinations comprise a GIP agonist peptide which exhibitsagonist activity at the GIP receptor and a glucagon antagonist peptidewhich exhibits antagonist or inhibitory activity at the glucagonreceptor. In specific aspects, the GIP agonist peptide exhibits at least0.1% activity (e.g., at least 0.5%, at least 0.75%, at least 1%, atleast 5%, at least 10%) of native GIP at the GIP receptor and theglucagon antagonist peptide exhibits at least 60% inhibition of themaximum response achieved by glucagon at the glucagon receptor. Inspecific aspects, the IC50 at the glucagon receptor of the glucagonantagonist peptide is within about 10-fold (higher or lower) of the EC50at the GIP receptor of the GIP agonist peptide. In exemplaryembodiments, either or both of the GIP agonist peptide and glucagonantagonist peptide additionally exhibit agonist activity at the GLP-1receptor.

In some embodiments, the peptide combinations are provided as acomposition, such as, for example, a pharmaceutical composition. In someaspects, the composition comprises the GIP agonist peptide in admixturewith the glucagon antagonist peptide and the two peptides are notattached to one another.

In some embodiments, the peptide combinations are provided as aconjugate in which the GIP agonist peptide is attached via covalent ornon-covalent bonds (or a mixture of both types of bonds) to the glucagonantagonist peptide. In certain embodiments, the GIP agonist peptide iscovalently attached to the glucagon antagonist peptide via peptidebonds. In some aspects, the conjugate is a single polypeptide chain(e.g., a fusion peptide) comprising the GIP agonist peptide and glucagonantagonist peptide. In specific aspects, the fusion peptide can beproduced recombinantly. In alternative aspects, the GIP agonist peptideis attached to the glucagon antagonist peptide via one or more sidechain functional groups of one or more amino acids of the GIP agonistpeptide and/or glucagon antagonist peptide. In some aspects, theconjugate is a heterodimer (or multimer) comprising the GIP agonistpeptide and glucagon antagonist peptide attached to one another. Inspecific aspects, such as any of the above aspects, the GIP agonistpeptide is attached to the glucagon antagonist peptide via a linker,e.g., a bifunctional linker. In some aspects, the bifunctional linker isa hydrophilic polymer, e.g., polyethylene glycol. In certain specificaspects, the bifunctional linker connects a Cys residue of one of theGIP agonist peptide and glucagon antagonist peptide to a Lys of theother peptide. In certain embodiments of the present disclosures, eachof the Cys and Lys is located at the C-terminus of the peptide or withinthe C-terminal region of the peptide.

In some embodiments, the peptide combination is provided as a kit. Insome aspects, the GIP agonist peptide is packaged together with theglucagon antagonist peptide. In alternative aspects, the GIP agonistpeptide is packaged separately from the glucagon antagonist peptide. Thekit in some aspects comprises instructions for administering the GIPagonist peptide and glucagon antagonist peptide. In some aspects, thekit comprises instructions for co-administering the GIP agonist peptideand glucagon antagonist peptide.

The present disclosures therefore provides compositions (e.g.,pharmaceutical compositions), conjugates (e.g., fusion peptides,heterodimers), and kits, each of which comprise a GIP agonist peptideand a glucagon antagonist peptide. Methods of using such compositions,conjugates, and kits are further provided herein. For example, thepresent disclosures provide a method of treating a metabolic disease(e.g., diabetes, obesity) in a patient, comprising administering to thepatient any of the compositions or conjugates described herein in anamount effective to treat the metabolic disease in the patient. Thetreatment of other diseases is further contemplated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the % change in body weight in mice as a functionof time (days) after administration of vehicle alone (closed uprighttriangles), GLP-1 E 16 agonist at 10 nmol/kg (closed inverted triangles)or 35 nmol/kg (open squares), triagonist peptide MT-170 at 10 nmol/kg(open inverted triangles) or 35 nmol/kg (closed diamonds), or GLP-1/GIPco-agonist peptide MT-178 at 10 nmol/kg (grey inverted triangles) or at35 nmol/kg (grey squares).

FIG. 2 is a graph of the change in blood glucose levels (mg/dL) in miceat Day 7 after administration of vehicle alone (black bar), GLP-1 E 16agonist at 10 nmol/kg (white bar) or 35 nmol/kg (grey bar), triagonistpeptide MT-170 at 10 nmol/kg (horizontal lined bar) or 35 nmol/kg(vertical lined bar), or GLP-1/GIP co-agonist peptide MT-178 at 10nmol/kg (right-left diagonal lined bar) or at 35 nmol/kg (left-rightdiagonal lined bar).

FIG. 3 represents a graph of the blood glucose levels (mg/dL) as afunction of time before and after a glucose injection (administered attimepoint 0) of mice injected (at timepoint −60) with a vehicle control,a GLP-1 agonist peptide control, a lactam-containing (cyclic),pegylated, GIP-active glucagon analog (“mt-178”), or a lactam-lacking(linear), pegylated, GIP-active glucagon analog (“mt-274”) at 1, 3, or10 nmol/kg/week. The data of this figure excludes the data of four mice,as these mice exhibited aggressive behavior and substantial weight loss.

FIG. 4 represents a graph of the blood glucose levels (mg/dL) as afunction of time before and after a glucose injection (administered attimepoint 0) of mice injected (24 hours before the glucose injection)with a vehicle control, a GLP-1 agonist peptide control, mt-178, ormt-274 at 1, 3, or 10 nmol/kg/week. The data of this figure excludes thedata of four mice, as these mice exhibited aggressive behavior andsubstantial weight loss.

FIG. 5 represents a graph of the blood glucose levels (mg/dL) of mice 0or 7 days after injection with a vehicle control, a GLP-1 agonistpeptide control, mt-178, or mt-274 at 1, 3, or 10 nmol/kg/week. The dataof this figure excludes the data of four mice, as these mice exhibitedaggressive behavior and substantial weight loss.

FIG. 6 represents a graph of the percent change in body weight of mice0, 1, 3, 5, and 7 days after injection with a vehicle control, a GLP-1agonist peptide control, mt-178, or mt-274 at 1, 3, or 10 nmol/kg/week.The data of this figure excludes the data of four mice, as these miceexhibited aggressive behavior and substantial weight loss.

FIG. 7 represents a graph of the blood glucose levels (mg/dL) of mice 0or 7 days after injection with a vehicle control, a GLP-1 agonistpeptide control, mt-178, mt-178(TE), mt-274, or mt-274(TE) at 10 or 35nmol/kg/week. “TE” indicates a PEG group attached to the Cys at position40.

FIG. 8 represents a graph of the change in blood glucose (mg/dL) of mice7 days after injection with a vehicle control, a GLP-1 agonist peptidecontrol, mt-178, mt-178(TE), mt-274, or mt-274(TE) at 10 or 35nmol/kg/week. “TE” indicates a PEG group attached to the Cys at position40.

FIG. 9 represents a graph of the percent change in body weight of mice0, 1, 3, 5, 7, and 10 days after injection with a vehicle control, aGLP-1 agonist peptide control, mt-178, mt-178(TE), mt-274, or mt-274(TE)at 10 or 35 nmol/kg/week. “TE” indicates a PEG group attached to the Cysat position 40.

FIG. 10 represents a graph of the percent change in body weight of mice7 days after injection with a vehicle control, a GLP-1 agonist peptidecontrol, mt-178, mt-178(TE), mt-274, or mt-274(TE) at 10 or 35nmol/kg/week. “TE” indicates a PEG group attached to the Cys at position40.

FIG. 11 represents a graph of the change in blood glucose levels (mg/dL)of mice 0 and 7 days after QD injections for 7 days with a vehiclecontrol, liraglutide (an acylated GLP-1 analog), a C14 fatty acylated,unpegylated linear peptide (“mt-260”), a C16 fatty acylated, unpegylatedlinear peptide (“mt-261”), or a C18 fatty acylated, unpegylated linearpeptide (“mt-262”) at 25 or 125 nmol/kg.

FIG. 12 represents a graph of the percent change in body weight of mice0, 1, 3, 5, and 7 days after injection with a vehicle control,liraglutide, mt-260, mt-261, or mt-262 at 25 or 125 nmol/kg.

FIG. 13 represents a graph of the percent change in body weight of mice7 days after injection with a vehicle control, liraglutide, mt-260,mt-261, or mt-262 at 25 or 125 nmol/kg.

FIG. 14 represents a graph of the change in body weight (g) of mice 0,1, 3, 5, and 7 days after the first injection with a vehicle control,liraglutide (30 nmol/kg/day), or mt-261 (0.3, 1, 3, 10, or 30nmol/kg/day).

FIG. 15 represents a graph of the fat mass of mice 7 days after thefirst injection with a vehicle control, liraglutide (30 nmol/kg/day), ormt-261 (0.3, 1, 3, 10, or 30 nmol/kg/day).

FIG. 16 represents a graph of the blood glucose levels (mg/dL) of mice 0and 7 days after the first injection with a vehicle control, liraglutide(30 nmol/kg/day), or mt-261 (0.3, 1, 3, 10, or 30 nmol/kg/day).

FIG. 17 represents a line graph of the change in body weight (% change)as a function of time of mice injected with mt-263, Exendin-4, or avehicle control at the doses (nmol/kg/day) indicated in ( ).

FIG. 18 represents a bar graph of the total change in body weight (%)(as measured on Day 7 in comparison to Day 0) of mice injected withmt-263, Exendin-4, or a vehicle control at the doses (nmol/kg/day)indicated in ( ).

FIG. 19 represents a bar graph of the change in blood glucose levels(mg/dL) (as measured on Day 7 in comparison to Day 0) of mice injectedwith mt-263, Exendin-4, or a vehicle control at the doses (nmol/kg/day)indicated in ( ).

FIG. 20 represents a graph of the % change in body weight of mice 0, 1,3, 5, and 7 days after the first injection with a vehicle control,liraglutide, mt-277, mt-278, or mt-279.

FIG. 21 represents a graph of the blood glucose levels (mg/dL) of mice 0and 7 days after the first injection with a vehicle control,liraglutide, mt-277, mt-278, or mt-279.

FIG. 22 represents a graph of the total change in body weight (%) ofmice as measured 7 days after administration of mt-331, mt-311, or avehicle control. Doses (nmol/kg) are indicated in ( ).

FIG. 23 represents a graph of the total food intake (g) by mice asmeasured 7 days after administration of mt-331, mt-311, or a vehiclecontrol. Doses (nmol/kg) are indicated in ( ).

FIG. 24 represents a graph of the total change in blood glucose levelsof mice as measured 7 days after administration of mt-331, mt-311, or avehicle control. Doses (nmol/kg) are indicated in ( ).

FIG. 25 represents a graph of the total change in body weight of mice asmeasured 7 days after administration of mt-331, mt-353, or a vehiclecontrol at the indicated dose (nmol/kg) shown in ( ).

FIG. 26 represents a graph of the total food intake (g) by mice asmeasured 7 days after administration of mt-331, mt-353, or a vehiclecontrol at the indicated dose (nmol/kg) shown in ( ).

FIG. 27 represents a graph of the change in blood glucose levels (mg/dL)of mice as measured 7 days after administration of mt-331, mt-353, or avehicle control at the indicated dose (nmol/kg) shown in ( ).

FIG. 28 represents a graph of the total change in body weight (%) ofmice as measured 7 days after the first administration of mt-277,mt-278, mt-279, or a vehicle control.

FIG. 29 represents a graph of the total change in body weight (%) ofmice as measured 6 days after the first administration of mt-261,mt-309, or a vehicle control.

FIG. 30 represents a graph of the blood glucose levels (mg/dL) of miceas measured 6 days after the first administration of mt-261, mt-309, ora vehicle control. The first bar of each pair of bars of the samepattern is the blood glucose levels as measured on Day 0 and the secondbar of each pair is the levels on Day 6.

FIG. 31 represents a bar graph of the total change in body weight (%) asmeasured 6 days after the first administration of mt-261 (in comparisonto the body weight as measured on the first day of administration) ofmice injected with a vehicle control or mt-261 as further describedherein.

FIG. 32 represents a graph of the total change in body weight (%) ofmice injected with different acylated peptides (MT-261, MT-367, MT-270,and MT-369) as calculated by subtracting the body weight on Day 0 fromthe body weight on Day 7.

FIG. 33 represents a graph of the total change in blood glucose levelsof mice injected with different acylated peptides (MT-261, MT-367,MT-270, and MT-369) as calculated by subtracting the blood glucoselevels on Day 0 from that on Day 7.

FIG. 34 represents a graph of the total change in body weight (%) ofmice injected with MT-367, MT-369, MT-368, MT-384, MT-385, or MT-364 ascalculated by subtracting the body weight on Day 0 from the body weighton Day 7.

FIG. 35 represents a graph of the total change in blood glucose levelsof mice injected with MT-367, MT-369, MT-368, MT-384, MT-385, or MT-364as calculated by subtracting the blood glucose levels on Day 0 from thaton Day 7.

FIG. 36 represents a graph of the change in body weight (%) of miceinjected with an Exendin-4-like peptide or with MT-263, MT-280, MT-356,or MT-357, or with a vehicle control, as a function of time (days).

FIG. 37 represents a graph of a graph of the blood glucose levels(ml/dL) of mice injected with an Exendin-4-like peptide or with MT-263,MT-280, MT-356, or MT-357, or with a vehicle control, as measured on Day0 and Day 7 of the study.

FIG. 38 represents a graph of the % change in body weight of miceinjected with vehicle only (daily or once every 3 days) or MT-263(daily, once every 2 days, or once every 3 days).

FIG. 39 represents a graph of the blood glucose levels as measured onDays 1 and 6 of the study of mice injected with vehicle only (daily oronce every 3 days) or MT-263 (daily, once every 2 days, or once every 3days).

FIG. 40 represents a graph of the total change in body weight observedin mice upon administration with acylated or pegylated compounds asfurther described herein.

FIG. 41 represents a graph of the change in blood glucose observed inmice upon administration with acylated or pegylated compounds as furtherdescribed herein.

FIG. 42 represents a graph of the total change in body weight observedin mice upon administration of MT-261 or MT-278 as further describedherein.

FIG. 43 represents a graph of the change in blood glucose observed inmice upon administration of MT-261 or MT-278 as further describedherein.

DETAILED DESCRIPTION OF THE PRESENT DISCLOSURES Definitions

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to altering glucose blood levels in the direction ofnormal levels and may include increasing or decreasing blood glucoselevels depending on a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hypoglycemia, asmeasured, for example, by an increase in blood glucose level. Analternative desired effect for the glucagon peptides of the presentdisclosure would include treating hyperglycemia, e.g., as measured by achange in blood glucose level closer to normal, or inducing weightloss/preventing weight gain, e.g., as measured by reduction in bodyweight, or preventing or reducing an increase in body weight, ornormalizing body fat distribution. The amount that is “effective” willvary from subject to subject, depending on the age and general conditionof the individual, mode of administration, and the like. Thus, it is notalways possible to specify an exact “effective amount.” However, anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route, e.g., subcutaneous, intramuscular, intraspinal, orintravenous.

The term “isolated” as used herein means having been removed from itsnatural environment. In some embodiments, the analog is made throughrecombinant methods and the analog is isolated from the host cell.

The term “purified,” as used herein relates to the isolation of amolecule or compound in a form that is substantially free ofcontaminants normally associated with the molecule or compound in anative or natural environment and means having been increased in purityas a result of being separated from other components of the originalcomposition. The term “purified polypeptide” is used herein to describea polypeptide which has been separated from other compounds including,but not limited to nucleic acid molecules, lipids and carbohydrates.

As used herein, the term “peptide” encompasses a sequence of 2 or moreamino acids and typically less than 50 amino acids, wherein the aminoacids are naturally occurring or coded or non-naturally occurring ornon-coded amino acids. Non-naturally occurring amino acids refer toamino acids that do not naturally occur in vivo but which, nevertheless,can be incorporated into the peptide structures described herein.“Non-coded” as used herein refer to an amino acid that is not anL-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp, Tyr.

As used herein, the terms “polypeptide” and “protein” are terms that areused interchangeably to refer to a polymer of amino acids, withoutregard to the length of the polymer. Typically, polypeptides andproteins have a polymer length that is greater than that of “peptides.”In some instances, a protein comprises more than one polypeptide chaincovalently or noncovalently attached to each other.

As used herein, a “peptide combination” encompasses a composition,conjugate, or kit comprising a GIP agonist peptide and a glucagonantagonist peptide. The GIP agonist peptide and a glucagon antagonistpeptide may be separated or mixed together, or may be linked covalentlyor non-covalently. When the peptides are linked covalently ornon-covalently, the peptide combination is referred to as a “conjugate.”

Throughout the application, all references to a particular amino acidposition by number (e.g., position 28) refer to the amino acid at thatposition in native glucagon (SEQ ID NO: 1) or the corresponding aminoacid position in any analogs thereof. For example, a reference herein to“position 28” would mean the corresponding position 27 for an analog ofglucagon in which the first amino acid of SEQ ID NO: 1 has been deleted.Similarly, a reference herein to “position 28” would mean thecorresponding position 29 for a analog of glucagon in which one aminoacid has been added before the N-terminus of SEQ ID NO: 1.

As used herein an “amino acid modification” refers to (i) a substitutionor replacement of an amino acid of SEQ ID NO: 1 with a different aminoacid (naturally-occurring or coded or non-coded ornon-naturally-occurring amino acid), (ii) an addition of an amino acid(naturally-occurring or coded or non-coded or non-naturally-occurringamino acid), to SEQ ID NO: 1 or (iii) a deletion of one or more aminoacids of SEQ ID NO: 1.

In some embodiments, the amino acid substitution or replacement is aconservative amino acid substitution, e.g., a conservative substitutionof the amino acid at one or more of positions 2, 5, 7, 10, 11, 12, 13,14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29. As used herein, the term“conservative amino acid substitution” is the replacement of one aminoacid with another amino acid having similar properties, e.g., size,charge, hydrophobicity, hydrophilicity, and/or aromaticity, and includesexchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negative-charged residues and their amides and esters:

-   -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;

III. Polar, positive-charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

In some embodiments, the amino acid substitution is not a conservativeamino acid substitution, e.g., is a non-conservative amino acidsubstitution.

As used herein the term “charged amino acid” refers to an amino acidthat comprises a side chain that is negative-charged (i.e.,de-protonated) or positive-charged (i.e., protonated) in aqueoussolution at physiological pH. For example negative-charged amino acidsinclude aspartic acid, glutamic acid, cysteic acid, homocysteic acid,and homoglutamic acid, whereas positive-charged amino acids includearginine, lysine and histidine. Charged amino acids include the chargedamino acids among the 20 coded amino acids, as well as atypical ornon-naturally occurring or non-coded amino acids.

As used herein the term “acidic amino acid” refers to an amino acid thatcomprises a second acidic moiety (other than the alpha carboxylic acidof the amino acid), including for example, a side chain carboxylic acidor sulfonic acid group.

As used herein a “sulfonic acid derivative of cysteine” refers tocompounds of the general structure:

wherein X₆ is C₁-C₄ alkyl, C₂-C₄ alkenyl or C₂-C₄ alkynyl.

The term “C₁-C_(n) alkyl” wherein n can be from 1 through 6, as usedherein, represents a branched or linear alkyl group having from one tothe specified number of carbon atoms. Typical C₁-C₆ alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The terms “C₂-C_(n) alkenyl” wherein n can be from 2 through 6, as usedherein, represents an olefinically unsaturated branched or linear grouphaving from 2 to the specified number of carbon atoms and at least onedouble bond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl (—CH₂—CH═CH₂), 1,3-butadienyl, (—CH═CHCH═CH₂),1-butenyl (—CH═CHCH₂CH₃), hexenyl, pentenyl, and the like.

The term “C₂-C_(n) alkynyl” wherein n can be from 2 to 6, refers to anunsaturated branched or linear group having from 2 to n carbon atoms andat least one triple bond. Examples of such groups include, but are notlimited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,and the like.

As used herein the term “pH stabilized glucagon antagonist” refers to aglucagon antagonist that exhibits superior stability and solubility,relative to native glucagon, in aqueous buffers in the broadest pH rangeused for pharmacological purposes.

As used herein, the term “selectivity” of a molecule for a firstreceptor relative to a second receptor refers to the following ratio:EC50 of the molecule at the second receptor divided by the EC50 of themolecule at the first receptor. For example, a molecule that has an EC50of 1 nM at a first receptor and an EC50 of 100 nM at a second receptorhas 100-fold selectivity for the first receptor relative to the secondreceptor.

As used herein the term “native glucagon” refers to a peptide consistingof the sequence of SEQ ID NO: 1 and the term “native GLP-1” is a genericterm that designates GLP-1(7-36) amide, GLP-1(7-37) acid or a mixture ofthose two compounds. As used herein, the term “native GIP” refers to apeptide consisting of SEQ ID NO: 2.

As used herein, “GIP potency” or “potency compared to native GIP” of amolecule refers to the ratio of the EC50 of the molecule at the GIPreceptor divided by the EC50 of native GIP at the GIP receptor.

As used herein, “glucagon potency” or “potency compared to nativeglucagon” of a molecule refers to the ratio of the EC50 of the moleculeat the glucagon receptor divided by the EC50 of native glucagon atglucagon receptor.

As used herein, “GLP-1 potency” or “potency compared to native GLP-1” ofa molecule refers to the ratio of the EC50 of the molecule at GLP-1receptor divided by the EC50 of native GLP-1 at GLP-1 receptor.

The term “GIP agonist peptide” refers to a compound that binds to andactivates downstream signaling of the GIP receptor. However, this termshould not be construed as limiting the compound to having activity atonly the GIP receptor. Rather, the GIP agonist peptides of the presentdisclosures may exhibit additional activities at other receptors, asfurther discussed herein. GIP agonist peptides, for example, may exhibitactivity (e.g., agonist activity) at the GLP-1 receptor. Also, the term“GIP agonist peptide” should not be construed as limiting the compoundto only peptides. Rather, compounds other than peptides are encompassedby this term. Accordingly, the GIP agonist peptide in some aspects is apeptide in conjugate form (a heterodimer, a multimer, a fusion peptide),a chemically-derivatized peptide, a pharmaceutical salt of a peptide, apeptidomimetic, and the like.

The term “glucagon antagonist peptide” refers to a compound thatcounteracts glucagon activity or prevents glucagon function. Forexample, a glucagon antagonist exhibits at least 60% inhibition (e.g.,at least 70% inhibition) and preferably, at least 80% inhibition, of themaximum response achieved by glucagon at the glucagon receptor. In oneembodiment, the glucagon antagonist exhibits at least 90% inhibition ofthe maximum response achieved by glucagon at the glucagon receptor. In aspecific embodiment, the glucagon antagonist exhibits 100% inhibition ofthe maximum response achieved by glucagon at the glucagon receptor.Additionally, a glucagon antagonist at a concentration of about 1 μMexhibits less than about 20% of the maximum agonist activity achieved byglucagon at the glucagon receptor. In one embodiment, the glucagonantagonist exhibits less than about 10% of the maximum agonist activityachieved by glucagon at the glucagon receptor. In a specific embodiment,the glucagon antagonist exhibits less than about 5% of the maximumagonist activity achieved by glucagon at the glucagon receptor. In yetanother specific embodiment, the glucagon antagonist exhibits 0% of themaximum agonist activity achieved by glucagon at the glucagon receptor.

The term “glucagon antagonist peptide” should not be construed aslimiting the compound to having activity at only the glucagon receptor.Rather, the glucagon antagonist peptides of the present disclosures mayexhibit additional activities at the glucagon receptor (e.g., partialagonism) or other receptor. Glucagon antagonist peptides, for example,may exhibit activity (e.g., agonist activity) at the GLP-1 receptor.Also, the term “glucagon antagonist peptide” should not be construed aslimiting the compound to only peptides. Rather, compounds other thanpeptides are encompassed by these terms. Accordingly, in some aspects,the GIP agonist peptide is a peptide in conjugate form, achemically-derivatized peptide, a pharmaceutical salt of a peptide, apeptidomimetic, and the like.

A “pure glucagon antagonist” is a glucagon antagonist that does notproduce any detected stimulation of glucagon or GLP-1 receptor activity,as measured by cAMP production using a validated in vitro model assay,such as that described in Example 2. For example, a pure glucagonantagonist exhibits less than about 5% (e.g., less than about 4%, lessthan about 3%, less than about 2%, less than about 1%, about 0%) of themaximum agonist activity achieved by glucagon at the glucagon receptorand exhibits less than about 5% (e.g., less than about 4%, less thanabout 3%, less than about 2%, less than about 1%, and about 0%) of themaximum agonist activity achieved by GLP-1 at the GLP-1 receptor.

EMBODIMENTS Peptide Combinations

The present disclosures provide peptide combinations comprising a GIPagonist peptide and a glucagon antagonist peptide. The activity of theGIP agonist peptide at the GIP receptor can be in accordance with any ofthe teachings set forth herein. Likewise, the activity of the glucagonantagonist peptide at the glucagon receptor can be in accordance withany of the teachings set forth herein. In specific aspects, the GIPagonist peptide exhibits at least 0.1% activity of native GIP at the GIPreceptor and the glucagon antagonist peptide exhibits at least 60%inhibition of the maximum response achieved by glucagon at the glucagonreceptor.

In specific aspects, the IC50 of the glucagon antagonist peptide at theglucagon receptor is within about 50-fold (e.g., within about 40-fold,within about 30-fold, within about 20-fold, within about 10-fold, withinabout 5-fold, within about 2-fold) of the EC50 at the GIP receptor ofthe GIP agonist peptide. In some embodiments, the EC50 at the GIPreceptor of the GIP agonist peptide is greater than the IC50 of theglucagon antagonist peptide at the glucagon peptide. In alternativeaspects, the EC50 at the GIP receptor of the GIP agonist peptide is lessthan the IC50 of the glucagon antagonist peptide at the glucagonpeptide. In certain aspects, the IC50 of the glucagon antagonist peptideat the glucagon receptor divided by the EC50 of the GIP agonist peptideat the GIP receptor is less than or about 100, 75, 60, 50, 40, 30, 20,15, 10, or 5, and no less than 1. In certain aspects, the EC50 of theGIP agonist peptide at the GIP receptor divided by the IC50 of theglucagon antagonist peptide at the glucagon receptor is less than orabout 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.

In exemplary embodiments, either or both of the GIP agonist peptide andglucagon antagonist peptide additionally exhibit agonist activity at theGLP-1 receptor. The activity at the GLP-1 receptor of either or both ofthe GIP agonist peptide and glucagon antagonist peptide may be inaccordance with any of the teachings described herein.

In some embodiments, the peptide combinations are provided as acomposition, such as, for example, a pharmaceutical composition. In someaspects, the GIP agonist peptide is in admixture with the glucagonantagonist peptide. The pharmaceutical composition in some aspectscomprises a pharmaceutical acceptable carrier.

In some embodiments, the peptide combinations are provided as aconjugate in which the GIP agonist peptide is attached via covalent ornon-covalent bonds (or a mixture of both types of bonds) to the glucagonantagonist peptide. In certain embodiments, the GIP agonist peptide iscovalently attached to the glucagon antagonist peptide via peptidebonds. In some aspects, the conjugate is a single polypeptide chain(e.g., a fusion peptide) comprising the GIP agonist peptide and glucagonantagonist peptide. In specific aspects, the fusion peptide can beproduced recombinantly. In alternative aspects, the GIP agonist peptideis attached to the glucagon antagonist peptide via one or more sidechain functional groups of one or more amino acids of the GIP agonistpeptide and/or glucagon antagonist peptide. In some aspects, theconjugate is a heterodimer (or multimer) comprising the GIP agonistpeptide and glucagon antagonist peptide attached to one another. Inspecific aspects, the GIP agonist peptide is attached to the glucagonantagonist peptide via a linker, e.g., a bifunctional linker. In someaspects, the bifunctional linker is a hydrophilic polymer, e.g.,polyethylene glycol. In certain specific aspects, the bifunctionallinker connects a Cys residue of one of the GIP agonist peptide andglucagon antagonist peptide to a Lys of the other peptide. In certainembodiments of the present disclosures, each of the Cys and Lys islocated at the C-terminus of the peptide or within the C-terminal regionof the peptide.

In some embodiments, the peptide combination is provided as a kit. Insome aspects, the GIP agonist peptide is packaged together with theglucagon antagonist peptide. In alternative aspects, the GIP agonistpeptide is packaged separately from the glucagon antagonist peptide. Thekit in some aspects comprises instructions of administering the GIPagonist peptide and glucagon antagonist peptide.

The GIP agonist peptide and glucagon antagonist peptide may beco-administered together or separately, simultaneously or sequentially(so long as both peptides exert the desired activity during anoverlapping time period). Methods of co-administering the GIP agonistpeptide and glucagon antagonist peptide for therapeutic purpose(s) areprovided herein. Also provided are the use of a GIP agonist peptide inthe preparation of a medicament for co-administration with a glucagonantagonist peptide and the use of a glucagon antagonist peptide in thepreparation of a medicament for co-administration with a GIP agonistpeptide.

Each of the foregoing peptide combinations comprising a GIP agonistpeptide and a glucagon antagonist peptide, as well as their methods ofuse, is further described herein.

Activity of the GIP Agonist Peptide

GIP Receptor Agonism

In some embodiments of the present disclosures, the GIP agonist peptideexhibits at least or about 0.1% activity of native GIP at the GIPreceptor. In exemplary embodiments, the GIP agonist peptide exhibits atleast or about 0.2%, at least or about 0.3%, at least or about 0.4%, atleast or about 0.5%, at least or about 0.6%, at least or about 0.7%, atleast or about 0.8%, at least or about 0.9%, at least or about 1%, atleast or about 5%, at least or about 10%, at least or about 20%, atleast or about 30%, at least or about 40%, at least or about 50%, atleast or about 60%, at least or about 70%, at least or about 75%, atleast or about 80%, at least or about 90%, at least or about 95%, or atleast or about 100% of the activity of native GIP at the GIP receptor.

In some embodiments of the present disclosures, the GIP agonist peptideexhibits activity at the GIP receptor which is greater than that ofnative GIP. In exemplary embodiments, the GIP agonist peptide exhibitsat least or about 101%, at least or about 105%, at least or about 110%,at least or about 125%, at least or about 150%, at least or about 175%at least or about 200%, at least or about 300%, at least or about 400%,at least or about 500% or higher % of the activity of native GIP at theGIP receptor. In some embodiments, the GIP agonist peptides describedherein exhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000%activity at the GIP receptor relative to native GIP. A peptide'sactivity at the GIP receptor relative to native GIP is calculated as theinverse ratio of EC50s for the GIP agonist peptide vs. native GIP. Insome embodiments, the GIP agonist peptide exhibits an EC50 for GIPreceptor activation which is in the nanomolar range. In exemplaryembodiments, the EC50 of the GIP agonist peptide at the GIP receptor isless than 1000 nM, less than 900 nM, less than 800 nM, less than 700 nM,less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM,less than 200 nM. In some embodiments, the EC50 of the peptide at theGIP receptor is about 100 nM or less, e.g., about 75 nM or less, about50 nM or less, about 25 nM or less, about 10 nM or less, about 8 nM orless, about 6 nM or less, about 5 nM or less, about 4 nM or less, about3 nM or less, about 2 nM or less, or about 1 nM or less. In someembodiments, the GIP agonist peptide exhibits an EC50 for GIP receptoractivation which is in the picomolar range. In exemplary embodiments,the EC50 of the GIP agonist peptide at the GIP receptor is less than1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than200 pM. In some embodiments, the EC50 of the peptide at the GIP receptoris about 100 pM or less, e.g., about 75 pM or less, about 50 pM or less,about 25 pM or less, about 10 pM or less, about 8 pM or less, about 6 pMor less, about 5 pM or less, about 4 pM or less, about 3 pM or less,about 2 pM or less, or about 1 pM or less. Receptor activation can bemeasured by in vitro assays measuring cAMP induction in HEK293 cellsover-expressing the GIP receptor, e.g. assaying HEK293 cellsco-transfected with DNA encoding the receptor and a luciferase genelinked to cAMP responsive element as described in Example 2.

Activity at the Glucagon Receptor

In many aspects of the present disclosures, the GIP agonist peptide doesnot activate the glucagon receptor to any appreciable degree.Accordingly, in some embodiments, the GIP agonist peptide is a GIPagonist which exhibits about 10% or less (e.g., about 9% or less, about8% or less, about 7% or less, about 6% or less, about 5% or less, about4% or less, about 3% or less, about 2% or less, about 1% or less) of theactivity of native glucagon at the glucagon receptor.

Co-Agonism

In some embodiments of the present disclosures, the GIP agonist peptideis a co-agonist peptide insofar as it activates a second receptordifferent from the GIP receptor, in addition to the GIP receptor. By wayof example, the GIP agonist peptide in some aspects exhibits activity atboth the GIP receptor and the GLP-1 receptor (“GLP-1/GIP receptorco-agonists”). In some embodiments, the EC50 of the GIP agonist peptideat the GIP receptor is within about 50- or less fold (higher or lower)than the EC50 of the GIP agonist peptide at the GLP-1 receptor. In someembodiments, the EC50 of the GIP agonist peptide at the GIP receptor iswithin about 40-fold, about 30-fold, about 20-fold (higher or lower)from its EC50 at the GLP-1 receptor. In some embodiments, the GIPpotency of the GIP agonist peptide is less than or about 25-, 20-, 15-,10-, or 5-fold different (higher or lower) from its GLP-1 potency. Insome embodiments, the ratio of the EC50 of the GIP agonist peptide atthe GIP receptor divided by the EC50 of the GIP agonist peptide at theGLP-1 receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10,or 5, and no less than 1. In some embodiments, the ratio of the GIPpotency of the GIP agonist peptide compared to the GLP-1 potency of theGIP agonist peptide is less than about 100, 75, 60, 50, 40, 30, 20, 15,10, or 5, and no less than 1. In some embodiments, the ratio of the EC50of the GIP agonist peptide at the GLP-1 receptor divided by the EC50 ofthe GIP agonist peptide at the GIP receptor is less than about 100, 75,60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In someembodiments, the ratio of the GLP-1 potency of the GIP agonist peptidecompared to the GIP potency of the GIP agonist peptide is less thanabout 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. Insome embodiments, the selectivity of the GIP agonist peptide does nothave at least 100-fold selectivity for the human GLP-1 receptor versusthe GIP receptor. In exemplary embodiments, the selectivity of the GIPagonist peptide for the human GLP-1 receptor versus the GIP receptor isless than 100-fold (e.g., less than or about 90-fold, less than or about80-fold, less than or about 70-fold, less than or about 60-fold, lessthan or about 50-fold, less than or about 40-fold, less than or about30-fold, less than or about 20-fold, less than or about 10-fold, lessthan or about 5-fold).

In some embodiments of the present disclosures, the GIP agonist peptideexhibits at least or about 0.1% activity of native GLP-1 at the GLP-1receptor. In exemplary embodiments, the GIP agonist peptide exhibits atleast or about 0.2%, at least or about 0.3%, at least or about 0.4%, atleast or about 0.5%, at least or about 0.6%, at least or about 0.7%, atleast or about 0.8%, at least or about 0.9%, at least or about 1%, atleast or about 5%, at least or about 10%, at least or about 20%, atleast or about 30%, at least or about 40%, at least or about 50%, atleast or about 60%, at least or about 70%, at least or about 75%, atleast or about 80%, at least or about 90%, at least or about 95%, or atleast or about 100% of the activity of native GLP-1 at the GLP-1receptor.

In some embodiments, the GIP agonist peptide exhibits activity at onlythe GIP receptor, and not the GLP-1 receptor. In some embodiments, theGIP agonist peptide is a GIP agonist which exhibits about 10% or less(e.g., about 9% or less, about 8% or less, about 7% or less, about 6% orless, about 5% or less, about 4% or less, about 3% or less, about 2% orless, about 1% or less) of the activity of native GLP-1 at the glucagonGLP-1.

Activity of Conjugates

In some embodiments, when the GIP agonist peptide is conjugated to aheterologous moiety (e.g., a hydrophilic moiety), as further describedherein, the GIP agonist peptide exhibits a decreased activity (e.g., alower potency or higher EC50) than when the GIP agonist peptide is in afree or unconjugated form. In some aspects, when the GIP agonist peptideis free or unconjugated, the GIP agonist peptide exhibits a potency atthe GIP receptor that is about 10-fold or greater than the potency ofthe GIP agonist peptide when the GIP agonist peptide is conjugated to aheterologous moiety (e.g., a hydrophilic moiety). In some aspects, whenunconjugated, the GIP agonist peptide exhibits a potency at the GIPreceptor that is about 10-fold, about 15-fold, about 20-fold, about25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold,about 50-fold, about 100-fold or more higher than the potency of the GIPagonist peptide when conjugated to a heterologous moiety. In someaspects, when unconjugated, the GIP agonist peptide exhibits a potencyat the GIP receptor that is about 10-fold, about 15-fold, about 20-fold,about 25-fold, about 30-fold, about 35-fold, about 40-fold, about45-fold, about 50-fold, about 100-fold or more higher than the potencyof the GIP agonist peptide when conjugated to a glucagon antagonistpeptide.

Structure of the GIP Agonist Peptide

Analogs of Native Human GIP

In some embodiments of the present disclosures, the GIP receptor agonistis an analog of native human GIP, the amino acid sequence of which isprovided herein as SEQ ID NO: 2. Accordingly, in some embodiments, theGIP agonist peptide comprises an amino acid sequence which is based onthe amino acid sequence of SEQ ID NO: 2 but is modified with 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in some instances, 16 or more(e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.), amino acidmodifications. In some embodiments, the GIP analog comprises a total of1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to9, or up to 10 amino acid modifications relative to the native human GIPsequence (SEQ ID NO: 2). In some embodiments, the modifications are anyof those described herein, e.g., acylation, alkylation, pegylation,truncation at C-terminus, substitution of the amino acid at one or moreof positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27,28, and 29. Exemplary GIP receptor agonists are known in the art. See,for example, Irwin et al., J Pharm and Expmt Ther 314(3): 1187-1194(2005); Salhanick et al., Bioorg Med Chem Lett 15(18): 4114-4117 (2005);Green et al., Diabetes 7(5): 595-604 (2005); O'Harte et al., JEndocrinol 165(3): 639-648 (2000); O'Harte et al., Diabetologia 45(9):1281-1291 (2002); Gault et al., Biochem J 367 (Pt3): 913-920 (2002);Gault et al., J Endocrin 176: 133-141 (2003); Irwin et al., DiabetesObes Metab. 11(6): 603-610 (epub 2009).

In some embodiments, the GIP agonist peptide of the present disclosurescomprises an amino acid sequence which has at least 25% sequenceidentity to the amino acid sequence of native human GIP (SEQ ID NO: 2).In some embodiments, the GIP agonist peptide comprises an amino acidsequence which is at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90% or hasgreater than 90% sequence identity to SEQ ID NO: 2. In some embodiments,the amino acid sequence of the GIP agonist peptide which has theabove-referenced % sequence identity is the full-length amino acidsequence of the GIP agonist peptide. In some embodiments, the amino acidsequence of the GIP agonist peptide which has the above-referenced %sequence identity is only a portion of the amino acid sequence of theGIP agonist peptide. In some embodiments, the GIP agonist peptidecomprises an amino acid sequence which has about A % or greater sequenceidentity to a reference amino acid sequence of at least 5 contiguousamino acids (e.g., at least 6, at least 7, at least 8, at least 9, atleast 10 amino acids) of SEQ ID NO: 2, wherein the reference amino acidsequence begins with the amino acid at position C of SEQ ID NO: 2 andends with the amino acid at position D of SEQ ID NO: 2, wherein A is 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99; C is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28 or 29. Any and all possible combinations of the foregoingparameters are envisioned, including but not limited to, e.g., wherein Ais 90% and C and D are 1 and 27, or 6 and 27, or 8 and 27, or and 27, or12 and 27, or 16 and 27.

In specific aspects, the GIP agonist peptide is an analog of native GIPcomprising an amino acid modification at position 1, position 2, or atboth positions 1 and 2, wherein the amino acid modification confers thepeptide with increased resistance to DPP-IV protease cleavage. In someaspects, the amino acid modification which confers the peptide withincreased resistance to DPP-IV protease cleavage are any of thosedescribed herein with regard to analogs of native human glucagon. Forexample, the amino acid modification may be a substitution of the Tyr atposition 1 of SEQ ID NO: 2 with an amino acid selected from the groupconsisting of D-histidine, alpha, alpha-dimethyl imidiazole acetic acid(DMIA), N-methyl histidine, alpha-methyl histidine, imidazole aceticacid, desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine. Alternatively or additionally, the amino acidmodification is a substitution of the Ala at position 2 of SEQ ID NO: 2with an amino acid selected from the group consisting of D-serine,D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, andaminoisobutyric acid (AIB). In some specific aspects, the GIP agonistpeptide which is an analog of native GIP comprises or further comprisesa C-terminal extension comprising 1-21 amino acids. Such extensions areknown in the art and include those described herein with regard tofusion peptides of glucagon analogs. In specific aspects, the extensioncomprises the amino acid sequence of any of SEQ ID NOs: 3 to 9. In someembodiments, the Xaa of SEQ ID NO: 4, 6, or 7 is a small, aliphaticresidue, e.g., a Gly. In some embodiments, the C-terminal extensioncomprises 1-6 positive-charged amino acids, e.g., Arg, an analog of Arg,an amino acid of Formula IV, e.g., Lys, d-Lys, Orn, Dab, etc. In someembodiments, GIP agonist peptide is an analog of GIP and comprises anamino acid modification which reduces agonist activity at the GIPreceptor to, e.g., a level such that the EC50 at the GIP receptor iswithin 10-fold of the IC50 of the glucagon antagonist peptide of thepeptide combination. Suitable amino acid modifications that reduce GIPagonist activity include, for example, substitution of the Tyr atposition 1 with a small aliphatic amino acid residue, e.g., Ala, Gly, orwith an imidazole containing amino acid, e.g., His, or an analogthereof. In some aspects, the amino acid modification that reduce GIPagonist activity is a deletion of the amino acid at position 1 or adeletion of the amino acids at positions 1 and 2. Additionalmodifications of the native GIP amino acid sequence (SEQ ID NO: 2), suchas any of those taught herein in the context of a GIP agonist peptidewhich is an analog of native glucagon, e.g., any of those taught asaffecting activity at the GIP receptor, GLP-1 receptor, and/or glucagonreceptor, increasing stability, solubility, half-life, time of action,and the like, are contemplated.

Analogs of Native Human Glucagon

In some embodiments, the GIP agonist peptide is structurally similar tonative human glucagon (SEQ ID NO: 1), e.g., is an analog of native humanglucagon (or “glucagon analog”). Such analogs of glucagon exhibiting GIPreceptor agonist activity are known in the art. See, for example, theteachings of International Patent Application No. PCT US2009/47447(filed on Jun. 16, 2009), U.S. Application No. 61/073,274 (filed Jun.17, 2008); U.S. Application No. 61/078,171 (filed Jul. 3, 2008); U.S.Application No. 61/090,448 (filed Aug. 20, 2008), U.S. Application No.61/151,349 (filed Feb. 10, 2009), and U.S. Application No. 61/187,578;the contents of which are incorporated by reference in their entirety.

In some embodiments, the GIP agonist peptide is an analog of nativehuman glucagon (SEQ ID NO: 1) which comprises an amino acid sequencebased on the amino acid sequence of SEQ ID NO: 1 but is modified with 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in some instances,16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.), amino acidmodifications. In some embodiments, the GIP agonist peptide comprises atotal of 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to8, up to 9, or up to 10 amino acid modifications relative to the nativehuman glucagon sequence (SEQ ID NO: 1). In some embodiments, themodifications are any of those described herein, e.g., acylation,alkylation, pegylation, truncation at C-terminus, substitution of theamino acid at one or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17,18, 19, 20, 21, 23, 24, 27, 28, and 29.

In some embodiments, the GIP agonist peptide of the present disclosurescomprises an amino acid sequence which has at least 25% sequenceidentity to the amino acid sequence of native human glucagon (SEQ ID NO:1). In some embodiments, the GIP agonist peptide comprises an amino acidsequence which is at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90% or hasgreater than 90% sequence identity to SEQ ID NO: 1. In some embodiments,the amino acid sequence of the GIP agonist peptide which has theabove-referenced % sequence identity is the full-length amino acidsequence of the GIP agonist peptide. In some embodiments, the amino acidsequence of the GIP agonist peptide which has the above-referenced %sequence identity is only a portion of the amino acid sequence of theGIP agonist peptide. In some embodiments, the GIP agonist peptidecomprises an amino acid sequence which has about A % or greater sequenceidentity to a reference amino acid sequence of at least 5 contiguousamino acids (e.g., at least 6, at least 7, at least 8, at least 9, atleast 10 amino acids) of SEQ ID NO: 1, wherein the reference amino acidsequence begins with the amino acid at position C of SEQ ID NO: 1 andends with the amino acid at position D of SEQ ID NO: 1, wherein A is 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99; C is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28 or 29. Any and all possible combinations of the foregoingparameters are envisioned, including but not limited to, e.g., wherein Ais 90% and C and D are 1 and 27, or 6 and 27, or 8 and 27, or and 27, or12 and 27, or 16 and 27.

The GIP agonist peptides which are analogs of native human glucagon (SEQID NO: 1) described herein may comprise a peptide backbone of any numberof amino acids, i.e., can be of any peptide length. In some embodiments,the GIP agonist peptides described herein are the same length as SEQ IDNO: 1, i.e., are 29 amino acids in length. In some embodiments, the GIPagonist peptide is longer than 29 amino acids in length, e.g., the GIPagonist peptide comprises a C-terminal extension of 1-21 amino acids, asfurther described herein. Accordingly, the GIP agonist peptide in someembodiments, is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 amino acids in length. In someembodiments, the GIP agonist peptide is up to 50 amino acids in length.In some embodiments, the GIP agonist peptide is longer than 29 aminoacids in length (e.g., greater than 50 amino acids, (e.g., at least orabout 60, at least or about 70, at least or about 80, at least or about90, at least or about 100, at least or about 150, at least or about 200,at least or about 250, at least or about 300, at least or about 350, atleast or about 400, at least or about 450, at least or about 500 aminoacids in length) due to fusion with another peptide. In otherembodiments, the GIP agonist peptide is less than 29 amino acids inlength, e.g., 28, 27, 26, 25, 24, 23, amino acids.

In accordance with the foregoing, in some aspects, the GIP agonistpeptide of the present disclosures is an analog of native human glucagon(SEQ ID NO: 1) comprising SEQ ID NO: 1 modified with one or more aminoacid modifications which affect GIP activity, glucagon activity, and/orGLP-1 activity, enhance stability, e.g., by reducing degradation of thepeptide (e.g., by improving resistance to DPP-IV proteases), enhancesolubility, increase half-life, delay the onset of action, extend theduration of action at the GIP, glucagon, or GLP-1 receptor, or acombination of any of the foregoing. Such amino acid modifications, inaddition to other modifications, are further described herein.

Exemplary Embodiments of the GIP Agonist Peptides which are GlucagonAnalogs

In accordance with some embodiments of the present disclosures, the GIPagonist peptide which is an analog of glucagon (SEQ ID NO: 1) comprisesSEQ ID NO: 1 with (a) an amino acid modification at position 1 thatconfers GIP agonist activity, (b) a modification which stabilizes thealpha helix structure of the C-terminal portion (amino acids 12-29) ofthe GIP agonist peptide, and (c) optionally, 1 to 10 (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10) further amino acid modifications. In someembodiments, the analog exhibits at least or about 0.1% (e.g., at leastor about 0.25%, at least or about 0.5%, at least or about 0.75%, atleast or about 1%) activity of native GIP at the GIP receptor or anyother activity level at the GIP receptor described herein.

In certain embodiments, the modification which stabilizes the alphahelix structure is one which provides or introduces an intramolecularbridge, including, for example, a covalent intramolecular bridge, suchas any of those described herein. The covalent intramolecular bridge insome embodiments is a lactam bridge. The lactam bridge of the GIPagonist peptide of these embodiments can be a lactam bridge as describedherein. See, e.g., the teachings of lactam bridges under the section“Stabilization of the Alpha Helix Structure.” For example, the lactambridge may be one which is between the side chains of amino acids atpositions i and i+4 or between the side chains of amino acids atpositions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, and whereinj is 17. In certain embodiments, the lactam bridge can be between theamino acids at positions 16 and 20, wherein one of the amino acids atpositions 16 and 20 is substituted with Glu and the other of the aminoacids at positions 16 and 20 is substituted with Lys.

In alternative embodiments, the modification which stabilizes the alphahelix structure is the introduction of one, two, three, or fourα,α-disubstituted amino acids at position(s) 16, 20, 21, and 24 of theGIP agonist peptide. In some embodiments, the α,α-disubstituted aminoacid is AIB. In certain aspects, the α,α-disubstituted amino acid (e.g.,AIB) is at position 20 and the amino acid at position 16 is substitutedwith a positive-charged amino acid, such as, for example, an amino acidof Formula IV, which is described herein. The amino acid of Formula IVmay be homoLys, Lys, Orn, or 2,4-diaminobutyric acid (Dab).

In any of the above exemplary embodiments, the amino acid modificationat position 1 that confers GIP agonist activity can be a substitution ofHis with an amino acid lacking an imidazole side chain. The amino acidmodification at position 1 can, for example, be a substitution of Hiswith a large, aromatic amino acid. In some embodiments, the large,aromatic amino acid is any of those described herein, including, forexample, Tyr.

In certain aspects in which the GIP agonist peptide comprises an aminoacid modification at position 1 that confers GIP agonist activity and amodification which stabilizes the alpha helix structure of theC-terminal portion (amino acids 12-29) of the GIP agonist peptide, theGIP agonist peptide further comprises amino acid modifications at one,two or all of positions 27, 28 and 29. In some aspects, the Met atposition 27 is substituted with a large aliphatic amino acid, optionallyLeu, the Asn at position 28 is substituted with a small aliphatic aminoacid, optionally Ala, the Thr at position 29 is substituted with a smallaliphatic amino acid, optionally Gly, or a combination of two or threeof the foregoing. In specific embodiments, the GIP agonist peptide whichis a glucagon analog comprises Leu at position 27, Ala at position 28,and Gly or Thr at position 29.

In certain embodiments of the present disclosures in which the GIPagonist peptide comprises an amino acid modification at position 1 thatconfers GIP agonist activity and a modification which stabilizes thealpha helix structure of the C-terminal portion (amino acids 12-29) ofthe GIP agonist peptide, the GIP agonist peptide further comprises anextension of 1 to 21 amino acids C-terminal to the amino acid atposition 29. The extension in some aspects comprises the amino acidsequence of SEQ ID NO: 3 or 4, for instance. Additionally oralternatively, the GIP agonist peptide in some aspects comprises anextension of which 1-6 amino acids of the extension are positive-chargedamino acids. The positive-charged amino acids in some embodiments areamino acids of Formula IV, including, but not limited to Lys, d-Lys,homoLys, Orn, and Dab. In some embodiments, the positive-charged aminoacid is Arg, or an analog thereof. In some aspects, the extensioncomprises 1-6 aa that are negative-charged amino acids, e.g., Asp, Glu.

In some embodiments in which the GIP agonist peptide comprises an aminoacid modification at position 1 that confers GIP agonist activity and amodification which stabilizes the alpha helix structure of theC-terminal portion (amino acids 12-29) of the GIP agonist peptide, theGIP agonist peptide is acylated or alkylated as described herein. Insome aspects, the acyl or alkyl group is attached to the GIP agonistpeptide, with or without a spacer, at position 10 or 40 of the GIPagonist peptide, as further described herein. The GIP agonist peptide inaddition or alternative aspects is modified to comprise a hydrophilicmoiety as further described herein. Furthermore, in some embodiments,the GIP agonist peptide comprises any one or a combination of thefollowing modifications:

-   -   (a) Ser at position 2 substituted with D-Ser, Ala, D-Ala, Gly,        N-methyl-Ser, AIB, Val, or α-amino-N-butyric acid;    -   (b) Tyr at position 10 substituted with Trp, Lys, Orn, Glu, Phe,        or Val:    -   (c) Linkage of an acyl group to a Lys at position 10;    -   (d) Lys at position 12 substituted with Arg or Ile;    -   (e) Ser at position 16 substituted with Glu, Gln, homoglutamic        acid, homocysteic acid, Thr, Gly, or AIB;    -   (f) Arg at position 17 substituted with Gln;    -   (g) Arg at position 18 substituted with Ala, Ser, Thr, or Gly;    -   (h) Gln at position 20 substituted with Ser, Thr, Ala, Lys,        Citrulline, Arg, Orn, or AIB;    -   (i) Asp at position 21 substituted with Glu, homoglutamic acid,        homocysteic acid;    -   (j) Val at position 23 substituted with Ile;    -   (k) Gln at position 24 substituted with Asn, Ser, Thr, Ala, or        AIB;    -   (l) and a conservative substitution at any of positions 2 5, 9,        10, 11, 12. 13, 14, 15, 16, 8 19 20, 21. 24, 27, 28, and 29.

In exemplary embodiments, the GIP agonist peptide which is an analog ofglucagon (SEQ ID NO: 1) comprises the following modifications:

-   -   (a) an amino acid modification at position 1 that confers GIP        agonist activity,    -   (b) a lactam bridge between the side chains of amino acids at        positions i and i+4 or between the side chains of amino acids at        positions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, and        wherein j is 17,    -   (c) amino acid modifications at one, two or all of positions 27,        28 and 29, e.g., amino acid modifications at position 27 and/or        28, and    -   (d) 1-9 or 1-6 further amino acid modifications, e.g. 1, 2, 3,        4, 5, 6, 7, 8 or 9 further amino acid modifications,        and the EC50 of the analog for GIP receptor activation is about        10 nM or less.

The lactam bridge of the GIP agonist peptide of these embodiments can bea lactam bridge as described herein. See, e.g., the teachings of lactambridges under the section “Stabilization of the Alpha Helix Structure.”For example, the lactam bridge can be between the amino acids atpositions 16 and 20, wherein one of the amino acids at positions 16 and20 is substituted with Glu and the other of the amino acids at positions16 and 20 is substituted with Lys.

In accordance with these embodiments, the analog can comprise, forexample, the amino acid sequence of any of SEQ ID NOs: 105-194. In someaspects, the GIP agonist peptide comprises a modified amino acidsequence of SEQ ID NOs: 105-194. in which the amino acid at position 1is substituted with Ala or is deleted.

In other exemplary embodiments, the GIP agonist peptide which is ananalog of glucagon (SEQ ID NO: 1) and which exhibits GIP agonistactivity comprises the following modifications:

-   -   (a) an amino acid modification at position 1 that confers GIP        agonist activity,    -   (b) one, two, three, or all of the amino acids at positions 16,        20, 21, and 24 of the analog is substituted with an        α,α-disubstituted amino acid,    -   (c) amino acid modifications at one, two or all of positions 27,        28 and 29, e.g., amino acid modifications at position 27 and/or        28, and    -   (d) 1-9 or 1-6 further amino acid modifications, e.g. 1, 2, 3,        4, 5, 6, 7, 8 or 9 further amino acid modifications,    -   and the EC50 of the analog for GIP receptor activation is about        10 nM or less.

The α,α-disubstituted amino acid of the GIP agonist peptide of theseembodiments can be any α,α-disubstituted amino acid, including, but notlimited to, amino iso-butyric acid (AIB), an amino acid disubstitutedwith the same or a different group selected from methyl, ethyl, propyl,and n-butyl, or with a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). In certain embodiments, theα,α-disubstituted amino acid is AIB. In certain embodiments, the aminoacid at position 20 is substituted with an α,α-disubstituted amino acid,e.g., AIB.

In accordance with these embodiments, the analog can comprise, forexample, the amino acid sequence of any of SEQ ID NOs: 199-241, 244-264,266-269, and 273-278. In some aspects, the GIP agonist peptide comprisesa modified amino acid sequence of SEQ ID NOs: 199-241, 244-264, 266-269,and 273-278 in which the amino acid at position 1 is substituted withAla or is deleted.

In yet other exemplary embodiments, the GIP agonist peptide which is ananalog of glucagon (SEQ ID NO: 1) comprises the following modifications:

-   -   (a) an amino acid modification at position 1 that confers GIP        agonist activity,    -   (b) an amino acid substitution of Ser at position 16 with an        amino acid of Formula IV:

wherein n is 1 to 16, or 1 to 10, or 1 to 7, or 1 to 6, or 2 to 6, eachof R1 and R2 is independently selected from the group consisting of H,C1-C18 alkyl, (C1-C18 alkyl)OH, (C1-C18 alkyl)NH2, (C1-C18 alkyl)SH,(C0-C4 alkyl)(C3-C6)cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocyclic),(C0-C4 alkyl)(C6-C10 aryl)R7, and (C1-C4 alkyl)(C3-C9 heteroaryl),wherein R7 is H or OH, and the side chain of the amino acid of FormulaIV comprises a free amino group,

-   -   (c) an amino acid substitution of the Gln at position 20 with an        alpha, alpha-disubstituted amino acid,    -   (d) amino acid modifications at one, two or all of positions 27,        28 and 29, e.g., amino acid modifications at position 27 and/or        28, and    -   (e) 1-9 or 1-6 further amino acid modifications, e.g. 1, 2, 3,        4, 5, 6, 7, 8 or 9 further amino acid modifications,        and the EC50 of the analog for GIP receptor activation is about        10 nM or less.

The amino acid of Formula IV of the analog of these embodiments may beany amino acid, such as, for example, the amino acid of Formula IV,wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.In certain embodiments, n is 2, 3, 4, or 5, in which case, the aminoacid is Dab, Orn, Lys, or homoLys respectively.

The alpha, alpha-disubstituted amino acid of the GIP agonist peptide ofthese embodiments may be any alpha, alpha-disubstituted amino acid,including, but not limited to, amino iso-butyric acid (AIB), an aminoacid disubstituted with the same or a different group selected frommethyl, ethyl, propyl, and n-butyl, or with a cyclooctane orcycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid). In certainembodiments, the alpha, alpha-disubstituted amino acid is AIB.

In accordance with these embodiments, the analog can comprise, forexample, the amino acid sequence of any of SEQ ID NOs: 199-265. In someaspects, the GIP agonist peptide comprises a modified amino acidsequence of SEQ ID NOs: 199-265 in which the amino acid at position 1 issubstituted with Ala or is deleted.

In yet other exemplary embodiments, the GIP agonist peptide which is ananalog of glucagon (SEQ ID NO: 1) comprises:

-   -   (a) an amino acid modification at position 1 that confers GIP        agonist activity, and    -   (b) an extension of about 1 to about 21 amino acids C-terminal        to the amino acid at position 29, wherein at least one of the        amino acids of the extension is acylated or alkylated,        wherein the EC50 of the analog for GIP receptor activation is        about 10 nM or less.

In some embodiments, the acylated or alkylated amino acid is an aminoacid of Formula I, II, or III. In more specific embodiments, the aminoacid of Formula I is Dab, Orn, Lys, or homoLys. Also, in someembodiments, the extension of about 1 to about 21 amino acids comprisesthe amino acid sequence of GPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQID NO: 4), wherein X is any amino acid, or GPSSGAPPPK (SEQ ID NO: 5) orXGPSSGAPPPK (SEQ ID NO: 6) or XGPSSGAPPPSK (SEQ ID NO: 7), wherein X isGly or a small, aliphatic or non-polar or slightly polar amino acid. Insome embodiments, the about 1 to about 21 amino acids may comprisesequences containing one or more conservative substitutions relative toSEQ ID NOs: 3, 4, 5, 6, or 7. In some embodiments, the acylated oralkylated amino acid is located at position 37, 38, 39, 40, 41, 42, or43 of the C-terminally-extended analog. In certain embodiments, theacylated or alkylated amino acid is located at position 40 of theC-terminally extended analog.

In some embodiments, the GIP agonist peptide which is an analog ofglucagon (SEQ ID NO: 1) further comprises amino acid modifications atone, two or all of positions 27, 28 and 29, e.g., amino acidmodifications at position 27 and/or 28.

In any of the above exemplary embodiments, the amino acid modificationat position 1 that confers GIP agonist activity can be a substitution ofHis with an amino acid lacking an imidazole side chain. The amino acidmodification at position 1 can, for example, be a substitution of Hiswith a large, aromatic amino acid. In some embodiments, the large,aromatic amino acid is any of those described herein, including, forexample, Tyr.

Also, with regard to the above exemplary embodiments, amino acidmodifications at one, two, or all of positions 27, 28, and 29 can be anyof the modifications at these positions described herein. For example,the Met at position 27 can be substituted with a large aliphatic aminoacid, optionally Leu, the Asn at position 28 can be substituted with asmall aliphatic amino acid, optionally Ala, and/or the Thr at position29 can be substituted with a small aliphatic amino acid, optionally Gly.Alternatively, the analog can comprise such amino acid modifications atposition 27 and/or 28.

The GIP agonist peptide of the above exemplary embodiments can furthercomprise 1-9 or 1-6 further, additional amino acid modifications, e.g.1, 2, 3, 4, 5, 6, 7, 8 or 9 further amino acid modifications, such as,for example, any of the modifications described herein which increase ordecrease the activity at any of the GIP, GLP-1, and glucagon receptors,improve solubility, improve duration of action or half-life incirculation, delay the onset of action, or increase stability. Theanalog can further comprise, for example, an amino acid modification atposition 12, optionally, a substitution with Ile, and/or amino acidmodifications at positions 17 and 18, optionally substitution with Q atposition 17 and A at position 18, and/or an addition of GPSSGAPPPS (SEQID NO: 3) or XGPSSGAPPPS (SEQ ID NO: 4), or sequences containing one ormore conservative substitutions relative to SEQ ID NO: 3 or 4, to theC-terminus. Accordingly, the GIP agonist peptide which is an analog ofglucagon (SEQ ID NO: 1) in some aspects comprises one or more of thefollowing modifications:

-   -   (a) Ser at position 2 substituted with D-Ser, Ala, D-Ala, Gly,        N-methyl-Ser, AIB, Val, or α-amino-N-butyric acid;    -   (b) Tyr at position 10 substituted with Trp, Lys, Orn, Glu, Phe,        or Val;    -   (c) Linkage of an acyl group to a Lys at position 10;    -   (d) Lys at position 12 substituted with Arg;    -   (e) Ser at position 16 substituted with Glu, Gln, homoglutamic        acid, homocysteic acid, Thr, Gly, or AIB;    -   (f) Arg at position 17 substituted with Gln;    -   (g) Arg at position 18 substituted with Ala, Ser, Thr, or Gly;    -   (h) Gln at position 20 substituted with Ala, Ser, Thr, Lys,        Citrulline, Arg, Orn, or AIB;    -   (i) Asp at position 21 substituted with Glu, homoglutamic acid,        homocysteic acid;    -   (j) Val at position 23 substituted with Ile;    -   (k) Gln at position 24 substituted with Asn, Ala, Ser, Thr, or        AIB; and    -   (l) a conservative substitution at any of positions 2, 5, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28, and 29.

The GIP agonist peptide in some embodiments comprises a combination ofthe modifications (a) through (l). Alternatively or additionally, theGIP agonist peptide can comprise an amino acid modification at position3 of SEQ ID NO: 1 (e.g., an amino acid substitution of Gln with Glu),wherein the GIP agonist peptide has less than 1% of the activity ofglucagon at the glucagon receptor. Alternatively or additionally, theGIP agonist peptide can comprise an amino acid modification at position7 of SEQ ID NO: 1 (e.g., an amino acid substitution of Thr with an aminoacid lacking a hydroxyl group, e.g., Abu or Ile), wherein the GIPagonist peptide has less than about 10% of the activity of GLP-1 at theGLP-1 receptor.

With regard to the exemplary embodiments, the GIP agonist peptide whichis an analog of glucagon (SEQ ID NO: 1) can be covalently linked to ahydrophilic moiety. In some embodiments, the GIP agonist peptide iscovalently linked to the hydrophilic moiety at any of amino acidpositions 16, 17, 20, 21, 24, 29, 40, or the C-terminus. In certainembodiments, the GIP agonist peptide comprises a C-terminal extension(e.g., an amino acid sequence of SEQ ID NO: 3) and an addition of anamino acid comprising the hydrophilic moiety, such that the hydrophilicmoiety is covalently linked to the GIP agonist peptide at position 40.

In some embodiments, the hydrophilic moiety is covalently linked to aLys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the GIP agonistpeptide. The Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine may bean amino acid that is native to the glucagon sequence (SEQ ID NO: 1) orit may be an amino acid which is replacing a native amino acid of SEQ IDNO: 1. In some embodiments, wherein the hydrophilic moiety is attachedto a Cys, the linkage to the hydrophilic moiety can comprise thestructure

With regard to the GIP agonist peptides comprising a hydrophilic moiety,the hydrophilic moiety may be any of those described herein. See, e.g.,the teachings under the section “Linkage of hydrophilic moieties.” Insome embodiments, the hydrophilic moiety is a polyethylene glycol (PEG).The PEG in certain embodiments has a molecular weight of about 1,000Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about40,000 Daltons.

With regard to any of the above exemplary embodiments, the GIP agonistpeptide which is an analog of glucagon (SEQ ID NO: 1) and which exhibitsGIP agonist activity in some embodiments comprises a modified amino acidin which the side chain is covalently linked to an acyl or alkyl group(e.g., an acyl or alkyl group which is non-native to anaturally-occurring amino acid). The acylated or alkylated analog can bein accordance with acylated or alkylated peptides described in thesection “Acylation and alkylation.” In some embodiments, the acyl groupis a C4 to a C30 fatty acyl group, such as, for example, a C10 fattyacyl or alkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acylor alkyl group, a C16 fatty acyl or alkyl group, a C18 fatty acyl oralkyl group, a C20 acyl or alkyl group, or a C22 acyl or alkyl group.The acyl or alkyl group may be covalently attached to any amino acid ofthe analog, including, but not limited to the amino acid at position 10or 40, or the C-terminal amino acid. In certain embodiments, the analogcomprises a C-terminal extension (e.g., an amino acid sequence of SEQ IDNO: 3) and an addition of an amino acid comprising the acyl or alkylgroup, such that the acyl or alkyl group is covalently linked to theanalog at position 40. In some embodiments, the acyl or alkyl group iscovalently linked to the side chain of an amino acid of Formula I, II,or III, e.g., a Lys residue. The acyl or alkyl group may be covalentlylinked to an amino acid which is native to the glucagon sequence (SEQ IDNO: 1) or may be linked to an amino acid which is added to the sequenceof SEQ ID NO: 1 or to the sequence of SEQ ID NO: 1 followed by SEQ IDNO: 3 (at the N- or C-terminus) or may be linked to an amino acid whichreplaces a native amino acid, e.g., the Tyr at position 10 of SEQ ID NO:1.

In the above exemplary embodiments, wherein the GIP agonist peptidecomprises an acyl or alkyl group, the GIP agonist peptide may beattached to the acyl or alkyl group via a spacer, as described herein.The spacer, for example, may be 3 to 10 atoms in length and may be, forinstance, an amino acid (e.g., 6-amino hexanoic acid, any amino aciddescribed herein), a dipeptide (e.g., Ala-Ala, βAla-βAla, Leu-Leu,Pro-Pro, γGlu-γGlu), a tripeptide, or a hydrophilic or hydrophobicbifunctional spacer. In certain aspects, the total length of the spacerand the acyl or alkyl group is about 14 to about 28 atoms.

In still further exemplary embodiments, the GIP agonist peptide which isan analog of glucagon (SEQ ID NO: 1) comprises the amino acid sequenceaccording to any one of SEQ ID NOs: 327, 328, 329, or 330 that furthercomprises the following modifications:

-   -   (a) optionally, an amino acid modification at position 1 that        confers GIP agonist activity,    -   (b) an extension of about 1 to about 21 amino acids C-terminal        to the amino acid at position 29, wherein at least one of the        amino acids of the extension is acylated or alkylated, and    -   (d) up to 6 further amino acid modifications,        wherein the EC50 of the analog for GIP receptor activation is        about 10 nM or less.

In some aspects, the GIP agonist peptide comprises SEQ ID NOs: 327-330in which the amino acid at position 1 is substituted with Ala or isdeleted.

In some aspects, the acylated or alkylated amino acid is an amino acidof Formula I, II, or III. In more specific embodiments, the amino acidof Formula I is Dab, Orn, Lys, or homoLys. Also, in some embodiments,the about 1 to about 21 amino acids comprises the amino acid sequence ofGPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ ID NO: 4), wherein X isany amino acid, or GPSSGAPPPK (SEQ ID NO: 5) or XGPSSGAPPPK (SEQ ID NO:6) or XGPSSGAPPPSK (SEQ ID NO: 7), wherein X is Gly or a small,aliphatic or non-polar or slightly polar amino acid. In someembodiments, the about 1 to about 21 amino acids may comprise sequencescontaining one or more conservative substitutions relative to SEQ ID NO:3, 4, 5, 6, or 7. In some embodiments, the acylated or alkylated aminoacid is located at position 37, 38, 39, 40, 41, 42, or 43 of theC-terminally-extended analog. In certain embodiments, the acylated oralkylated amino acid is located at position 40 of the C-terminallyextended analog.

In any of the above exemplary embodiments, the amino acid at position 1that confers GIP agonist activity can be an amino acid lacking animidazole side chain. The amino acid at position 1 can, for example, bea large, aromatic amino acid. In some embodiments, the large, aromaticamino acid is any of those described herein, including, for example,Tyr.

The GIP agonist peptide of the above exemplary embodiments can furthercomprise 1-6 further amino acid modifications, such as, for example, anyof the modifications described herein which increase or decrease theactivity at any of the GIP, GLP-1, and glucagon receptors, improvesolubility, improve duration of action or half-life in circulation,delay the onset of action, or increase stability.

In certain aspects, GIP agonist peptides described in the aboveexemplary embodiment, comprise further amino acid modifications at one,two or all of positions 27, 28 and 29. Modifications at these positionscan be any of the modifications described herein relative to thesepositions. For example, relative to SEQ ID NOs: 327, 328, 329, or 330,position 27 can be substituted with a large aliphatic amino acid (e.g.,Leu, Ile or norleucine) or Met, position 28 can be substituted withanother small aliphatic amino acid (e.g., Gly or Ala) or Asn, and/orposition 29 can be substituted with another small aliphatic amino acid(e.g., Ala or Gly) or Thr. Alternatively, the analog can comprise suchamino acid modifications at position 27 and/or 28.

The analog can further comprise one or more of the following additionalmodifications:

-   -   (a) the amino acid at position 2 is any one of D-Ser, Ala,        D-Ala, Gly, N-methyl-Ser, AIB, Val, or α-amino-N-butyric acid;    -   (b) the amino acid at position 10 is Tyr, Trp, Lys, Orn, Glu,        Phe, or Val;    -   (c) linkage of an acyl group to a Lys at position 10;    -   (d) the amino acid at position 12 is Ile, Lys or Arg;    -   (e) the amino acid at position 16 is any one of Ser, Glu, Gln,        homoglutamic acid, homocysteic acid, Thr, Gly, or AIB;    -   (f) the amino acid at position 17 is Gln or Arg;    -   (g) the amino acid at position 18 is any one of Ala, Arg, Ser,        Thr, or Gly;    -   (h) the amino acid at position 20 is any one of Ala, Ser, Thr,        Lys, Citrulline, Arg, Orn, or AIB or another alpha,        alpha-disubstituted amino acid;    -   (i) the amino acid at position 21 is any one of Glu, Asp,        homoglutamic acid, homocysteic acid;    -   (j) the amino acid at position 23 is Val or Ile;    -   (k) the amino acid at position 24 is any one of Gln, Asn, Ala,        Ser, Thr, or AIB; and    -   (l) one or more conservative substitutions at any of positions        2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27,        28, and 29.

The GIP agonist peptide in some embodiments comprises a combination ofthe modifications (a) through (l). Alternatively or additionally, theGIP agonist peptide can comprise an amino acid modification at position3 of SEQ ID NO: 1 (e.g., an amino acid substitution of Gln with Glu),wherein the GIP agonist peptide has less than 1% of the activity ofglucagon at the glucagon receptor. Alternatively or additionally, theGIP agonist peptide can comprise an amino acid modification at position7 of SEQ ID NO: 1 (e.g., an amino acid substitution of Thr with an aminoacid lacking a hydroxyl group, e.g., Abu or Ile), wherein the GIPagonist peptide has less than about 10% of the activity of GLP-1 at theGLP-1 receptor.

With regard to the exemplary embodiments, the analog can be covalentlylinked to a hydrophilic moiety. In some embodiments, the analog iscovalently linked to the hydrophilic moiety at any of amino acidpositions 16, 17, 20, 21, 24, 29, 40, or the C-terminus. In certainembodiments, the analog comprises a hydrophilic moiety covalently linkedto the analog at position 24.

In some embodiments, the hydrophilic moiety is covalently linked to aLys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog. TheLys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an aminoacid that is native to SEQ ID NO: 1, 227, 228, 229 or 230 of SequenceListing 2, or it may be a substituted amino acid. In some embodiments,wherein the hydrophilic moiety is linked to a Cys, the linkage maycomprise the structure

With regard to the GIP agonist peptides comprising a hydrophilic moiety,the hydrophilic moiety may be any of those described herein. See, e.g.,the teachings under the section “Linkage of hydrophilic moieties.” Insome embodiments, the hydrophilic moiety is a polyethylene glycol (PEG).The PEG in certain embodiments has a molecular weight of about 1,000Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about40,000 Daltons.

With regard to the exemplary embodiments, the GIP agonist peptide cancomprise a modified amino acid within the C-terminal extension in whichthe side chain is covalently linked to an acyl or alkyl group. Theacylated or alkylated analog can be in accordance with acylated oralkylated peptides described in the section “Acylation and alkylation.”In some embodiments, the acyl group is a C4 to a C30 fatty acyl group,such as, for example, a C10 fatty acyl or alkyl group, a C12 fatty acylor alkyl group, a C14 fatty acyl or alkyl group, a C16 fatty acyl oralkyl group, a C18 fatty acyl or alkyl group, a C20 acyl or alkyl group,or a C22 acyl or alkyl group. The acyl or alkyl group may be covalentlyattached to any amino acid of the analog, including, but not limited tothe amino acid at position 10 or 40, or the C-terminal amino acid. Insome embodiments, the acyl or alkyl group is covalently linked to theside chain of an amino acid of Formula I, II, or III, e.g., a Lysresidue. The acyl or alkyl group is covalently linked to an amino acidwhich is native to SEQ ID NO: 1, 327, 328, 329, or 330 or it may belinked to a substituted amino acid. The acyl or alkyl group iscovalently linked to an amino acid which is native to SEQ ID NO: 3, 4, 6or 7, or it may be linked to a substituted amino acid.

In the above exemplary embodiments, wherein the GIP agonist peptidecomprises an acyl or alkyl group, the GIP agonist peptidemay be attachedto the acyl or alkyl group via a spacer, as described herein. Thespacer, for example, may be 3 to 10 atoms in length and may be, forinstance, an amino acid (e.g., 6-amino hexanoic acid, any amino aciddescribed herein), a dipeptide (e.g., Ala-Ala, βAla-βAla, Leu-Leu,Pro-Pro, γGlu-γGlu), a tripeptide, or a hydrophilic or hydrophobicbifunctional spacer. In certain aspects, the total length of the spacerand the acyl or alkyl group is about 14 to about 28 atoms.

In some very specific embodiments, an GIP agonist peptide of the presentdisclosures comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 199-241, 244-264, 266, 292-307, 309-321 and323 or selected from the group consisting of SEQ ID NOs: 267-269,273-278, and 325. In some aspects, the GIP agonist peptide comprises amodified amino acid sequence of any of SEQ ID NOs: 199-241, 244-264,266, 292-307, 309-321, and 323 or any of SEQ ID NOs: 267-269, 273-278,and 325, in which the amino acid at position 1 is substituted with Alaor is deleted.

Further, specific examples of GIP agonist peptides of the presentdisclosures include but are not limited to, any of SEQ ID NOs: 105-194,199-246, 248-250, and 253-278.

In still further exemplary embodiments, the GIP agonist peptide which isan analog of glucagon (SEQ ID NO: 1) comprises an acyl or alkyl group(e.g., an acyl or alkyl group which is non-native to a naturallyoccurring amino acid), wherein the acyl or alkyl group is attached to aspacer, wherein (i) the spacer is attached to the side chain of theamino acid at position 10 of the analog; or (ii) the analog comprises anextension of 1 to 21 amino acids C-terminal to the amino acid atposition 29 and the spacer is attached to the side chain of an aminoacid corresponding to one of positions 37-43 relative to SEQ ID NO: 1,wherein the EC50 of the analog for GIP receptor activation is about 10nM or less.

In such embodiments, the GIP agonist peptide may comprise an amino acidsequence of SEQ ID NO: 1 with (i) an amino acid modification at position1 that confers GIP agonist activity, (ii) amino acid modifications atone, two, or all of positions 27, 28, and 29, (iii) at least one of:

-   -   (A) the analog comprises a lactam bridge between the side chains        of amino acids at positions i and i+4 or between the side chains        of amino acids at positions j and j+3, wherein i is 12, 13, 16,        17, 20 or 24, and wherein j is 17;    -   (B) one, two, three, or all of the amino acids at positions 16,        20, 21, and 24 of the analog is substituted with an        α,α-disubstituted amino acid; or    -   (C) the analog comprises (i) an amino acid substitution of Ser        at position 16 with an amino acid of Formula IV:

wherein n is 1 to 7, wherein each of R1 and R2 is independently selectedfrom the group consisting of H, C1-C18 alkyl, (C1-C18 alkyl)OH, (C1-C18alkyl)NH₂, (C1-C18 alkyl)SH, (C0-C4 alkyl)(C3-C6)cycloalkyl, (C0-C4alkyl)(C2-C5 heterocyclic), (C0-C4 alkyl)(C6-C10 aryl)R7, and (C1-C4alkyl)(C3-C9 heteroaryl), wherein R7 is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group; and (ii) anamino acid substitution of the Gln at position 20 with an alpha,alpha-disubstituted amino acid, and (iii) up to 6 further amino acidmodifications.

The alpha, alpha-disubstituted amino acid of the GIP agonist peptide ofthese embodiments may be any alpha, alpha-disubstituted amino acid,including, but not limited to, amino iso-butyric acid (AIB), an aminoacid disubstituted with the same or a different group selected frommethyl, ethyl, propyl, and n-butyl, or with a cyclooctane orcycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid). In certainembodiments, the alpha, alpha-disubstituted amino acid is AIB.

The amino acid of Formula IV of the GIP agonist peptide of theseembodiments may be any amino acid, such as, for example, the amino acidof Formula IV, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 16. In certain embodiments, n is 2, 3, 4, or 5, in whichcase, the amino acid is Dab, Orn, Lys, or homoLys respectively.

In any of the above exemplary embodiments, the amino acid modificationat position 1 that confers GIP agonist activity can be a substitution ofHis with an amino acid lacking an imidazole side chain. The amino acidmodification at position 1 can, for example, be a substitution of Hiswith a large, aromatic amino acid. In some embodiments, the large,aromatic amino acid is any of those described herein, including, forexample, Tyr.

Also, with regard to the above exemplary embodiments, amino acidmodifications at one, two, or all of positions 27, 28, and 29 can be anyof the modifications at these positions described herein. For example,the Met at position 27 can be substituted with a large aliphatic aminoacid, optionally Leu, the Asn at position 28 can be substituted with asmall aliphatic amino acid, optionally Ala, and/or the Thr at position29 can be substituted with a small aliphatic amino acid, optionally Gly.Alternatively, the GIP agonist peptide can comprise such amino acidmodifications at position 27 and/or 28.

The GIP agonist peptide of the above exemplary embodiments can furthercomprise 1-9 or 1-6 further, additional amino acid modifications, e.g.1, 2, 3, 4, 5, 6, 7, 8 or 9 further amino acid modifications, such as,for example, any of the modifications described herein which increase ordecrease the activity at any of the GIP, GLP-1, and glucagon receptors,improve solubility, improve duration of action or half-life incirculation, delay the onset of action, or increase stability. The GIPagonist peptide can further comprise, for example, an amino acidmodification at position 12, optionally, a substitution with Ile, and/oramino acid modifications at positions 17 and 18, optionally substitutionwith Q at position 17 and A at position 18, and/or an addition ofGPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ ID NO: 4), or sequencescontaining one or more conservative substitutions relative to SEQ ID NO:3 or 4, to the C-terminus. Accordingly, in some aspects, the GIP agonistpeptide which is an analog of glucagon (SEQ ID NO: 1) comprises one ormore of the following modifications:

-   -   (a) Ser at position 2 substituted with D-Ser, Ala, D-Ala, Gly,        N-methyl-Ser, AIB, Val, or α-amino-N-butyric acid;    -   (b) Tyr at position 10 substituted with Trp, Lys, Orn, Glu, Phe,        or Val;    -   (c) Linkage of an acyl group to a Lys at position 10;    -   (d) Lys at position 12 substituted with Arg;    -   (e) Ser at position 16 substituted with Glu, Gln, homoglutamic        acid, homocysteic acid, Thr, Gly, Lys, or AIB;    -   (f) Arg at position 17 substituted with Gln;    -   (g) Arg at position 18 substituted with Ala, Ser, Thr, or Gly;    -   (h) Gln at position 20 substituted with Ala, Ser, Thr, Lys,        Citrulline, Arg, Orn, or AIB;    -   (i) Asp at position 21 substituted with Glu, homoglutamic acid,        homocysteic acid;    -   (j) Val at position 23 substituted with Ile;    -   (k) Gln at position 24 substituted with Asn, Ala, Ser, Thr, or        AIB; and    -   (l) a conservative substitution at any of positions 2, 5, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28, and 29.

The GIP agonist peptide in some embodiments comprise a combination ofthe modifications (a) through (l). Alternatively or additionally, theGIP agonist peptide can comprise an amino acid modification at position3 of SEQ ID NO: 1 (e.g., an amino acid substitution of Gln with Glu),wherein the GIP agonist peptide has less than 1% of the activity ofglucagon at the glucagon receptor. Alternatively or additionally, theGIP agonist peptide can comprise an amino acid modification at position7 of SEQ ID NO: 1 (e.g., an amino acid substitution of Thr with an aminoacid lacking a hydroxyl group, e.g., Abu or Ile), a deletion of theamino acid(s) C-terminal to the amino acid at position 27 or 28,yielding a 27- or 28-amino acid peptide, or a combination thereof,wherein the GIP agonist peptide has less than about 10% of the activityof GLP-1 at the GLP-1 receptor.

With regard to the exemplary embodiments, the GIP agonist peptide can becovalently linked to a hydrophilic moiety. In some embodiments, the GIPagonist peptide is covalently linked to the hydrophilic moiety at any ofamino acid positions 16, 17, 20, 21, 24, 29, 40, or the C-terminus. Incertain embodiments, the GIP agonist peptide comprises a C-terminalextension (e.g., an amino acid sequence of SEQ ID NO: 3) and an additionof an amino acid comprising the hydrophilic moiety, such that thehydrophilic moiety is covalently linked to the GIP agonist peptide atposition 40.

In some embodiments, the hydrophilic moiety is covalently linked to aLys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the GIP agonistpeptide. The Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine may bean amino acid that is native to the glucagon sequence (SEQ ID NO: 1) orit may be an amino acid which is replacing a native amino acid of SEQ IDNO: 1. In some embodiments, wherein the hydrophilic moiety is attachedto a Cys, the linkage to the hydrophilic moiety can comprise thestructure

With regard to the GIP agonist peptides comprising a hydrophilic moiety,the hydrophilic moiety may be any of those described herein. See, e.g.,the teachings under the section “Linkage of hydrophilic moieties.” Insome embodiments, the hydrophilic moiety is a polyethylene glycol (PEG).The PEG in certain embodiments has a molecular weight of about 1,000Daltons to about 40,000 Daltons, e.g., about 20,000 Daltons to about40,000 Daltons.

In the exemplary embodiments, wherein the GIP agonist peptide comprisesan acyl or alkyl group, which is attached to the analog via a spacer,the spacer can be any spacer as described herein. The spacer, forexample, may be 3 to 10 atoms in length and may be, for instance, anamino acid (e.g., 6-amino hexanoic acid, any amino acid describedherein), a dipeptide (e.g., Ala-Ala, βAla-βAla, Leu-Leu, Pro-Pro,γGlu-γGlu), a tripeptide, or a hydrophilic or hydrophobic bifunctionalspacer. In certain aspects, the total length of the spacer and the acylor alkyl group is about 14 to about 28 atoms.

The acyl or alkyl group is any acyl or alkyl group as described herein,such as an acyl or alkyl group which is non-native to a naturallyoccurring amino acid. The acyl or alkyl group in some embodiments is aC4 to C30 fatty acyl group, such as, for example, a C10 fatty acyl oralkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acyl or alkylgroup, a C16 fatty acyl or alkyl group, a C18 fatty acyl or alkyl group,a C20 acyl or alkyl group, or a C22 acyl or alkyl group, or a C4 to C30alkyl group. In specific embodiments, the acyl group is a C12 to C18fatty acyl group (e.g., a C14 or C16 fatty acyl group).

In some embodiments, the extension of about 1 to about 21 amino acidsC-terminal to the amino acid at position 29 of the GIP agonist peptidecomprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 3) orXGPSSGAPPPS (SEQ ID NO: 4), wherein X is any amino acid, or GPSSGAPPPK(SEQ ID NO: 5) or XGPSSGAPPPK (SEQ ID NO: 6) or XGPSSGAPPPSK (SEQ ID NO:7), wherein X is Gly or a small, aliphatic or non-polar or slightlypolar amino acid. In some embodiments, the about 1 to about 21 aminoacids may comprise sequences containing one or more conservativesubstitutions relative to any of SEQ ID NO: 3, 4, 5, 6, or 7. In someembodiments, the acylated or alkylated amino acid is located at position37, 38, 39, 40, 41, 42, or 43 of the C-terminally-extended GIP agonistpeptide. In certain embodiments, the acylated or alkylated amino acid islocated at position 40 of the C-terminally extended GIP agonist peptide.

In some embodiments, the GIP agonist peptide which is an analog ofglucagon (SEQ ID NO: 1) is a peptide comprising the amino acid sequenceof any of the amino acid sequences, e.g., SEQ ID NOs: 105-194,optionally with up to 1, 2, 3, 4, or 5 further modifications that retainGIP agonist activity. In certain embodiments, the GIP agonist peptidewhich is an analog of glucagon (SEQ ID NO: 1) and which exhibits GIPagonist activity comprises the amino acids of any of SEQ ID NOs:199-362. In some aspects, the GIP agonist peptide comprises a modifiedamino acid sequence of SEQ ID NO: 199-362 in which the amino acid atposition 1 is substituted with Ala or is deleted.

In some embodiments of the present disclosures, the GIP agonist peptidecomprises the amino acid sequence of SEQ ID NO: 1 with at least oneamino acid modification (optionally, up to 15 amino acid modifications),and an extension of 1 to 21 amino acids C-terminal to the amino acid atposition 29 of the GIP agonist peptide. In certain aspects, the GIPagonist peptide comprises at least one amino acid modification and up to15 amino acid modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 amino acid modifications, up to 10 amino acidmodifications). In certain embodiments, the GIP agonist peptidescomprise at least one amino acid modification at up to 10 amino acidmodifications and additional conservative amino acid modifications.Conservative amino acid modifications are described herein.

In some aspects, at least one of the amino acid modifications confers astabilized alpha helix structure in the C-terminal portion of the GIPagonist peptide. Modifications which achieve a stabilized alpha helixstructure are described herein. See, for example, the teachings underthe section entitled Stabilization of the alpha helix. In some aspects,the GIP agonist peptide comprises an intramolecular bridge (e.g., acovalent intramolecular bridge, a non-covalent intramolecular bridge)between the side chains of two amino acids of the GIP agonist peptide.In certain aspects, an intramolecular bridge links the side chains ofthe amino acids at positions i and i+4, wherein i is 12, 13, 16, 17, 20,or 24. In other aspects, an intramolecular bridge connects the sidechains of the amino acids at positions j and j+3, wherein j is 17, or atpositions k and k+7″ wherein k is any integer between 12 and 22. Incertain embodiments, the intramolecular bridge is a covalentintramolecular bridge, e.g., a lactam bridge. In specific aspects, thelactam bridge connects the side chains of the amino acids at positions16 and 20. In particular aspects, one of the amino acids at positions 16and 20 is a positive-charged amino acid and the other is anegative-charged amino acid. For example, the GIP agonist peptide cancomprise a lactam bridge connecting the side chains of a Glu at position16 and a Lys at position 20. In other aspects, the negative-chargedamino acid and the positive-charged amino acid form a salt bridge. Inthis instance, the intramolecular bridge is a non-covalentintramolecular bridge.

In particular aspects, the amino acid modification which confers astabilized alpha helix is an insertion or substitution of an amino acidof SEQ ID NO: 1 with an α,α-disubstituted amino acid. Suitableα,α-disubstituted amino acids for purposes of stabilizing the alphahelix are described herein and include, for example, AIB. In someaspects, one, two, three, or more of the amino acids at positions 16,20, 21, and 24 of SEQ ID NO: 1 are substituted with an α,α-disubstitutedamino acid, e.g., AIB. In particular embodiments, the amino acid atposition 16 is AIB.

The GIP agonist peptide in some aspects comprises additionalmodifications, such as any of those described herein. For instance, theamino acid modifications may increase or decrease activity at the GLP-1receptor or decrease activity at the glucagon receptor. The amino acidmodifications may increase stability of the peptide, e.g., increaseresistance to DPP-IV protease degradation, stabilize the bond betweenamino acids 15 and 16. The amino acid modifications may increase thesolubility of the peptide and/or alter the time of action of the GIPagonist peptide at any of the GIP, glucagon, and GLP-1 receptors. Acombination of any of these types of modifications may be present in theGIP agonist peptides which exhibit agonist activity at the GIP receptor.

Accordingly, in some aspects, the GIP agonist peptide comprises theamino acid sequence of SEQ ID NO: 1 with one or more of: Gln at position17, Ala at position 18, Glu at position 21, Ile at position 23, and Ala,Asn, or Cys at position 24, or conservative amino acid substitutionsthereof. In some aspects, the GIP agonist peptide comprises a C-terminalamide in place of the C-terminal alpha carboxylate. In certainembodiments, the GIP agonist peptide comprises an amino acidsubstitution at position 1, position 2, or positions 1 and 2, whichsubstitution(s) achieve DPP-IV protease resistance. Suitable amino acidsubstitutions are described herein. For example, DMIA at position 1and/or d-Ser or AIB at position 2.

Additionally or alternatively, the GIP agonist peptide may comprise oneor a combination of: (a) Ser at position 2 substituted with Ala; (b) Glnat position 3 substituted with Glu or a glutamine GIP agonist peptide;(c) Thr at position 7 substituted with a Ile; (d) Tyr at position 10substituted with Trp or an amino acid comprising an acyl or alkyl groupwhich is non-native to a naturally-occurring amino acid; (e) Lys atposition 12 substituted with Ile; (f) Asp at position 15 substitutedwith Glu; (g) Ser at position 16 substituted with Glu; (h) Gln atposition 20 substituted with Ser, Thr, Ala, AIB; (i) Gln at position 24substituted with Ser, Thr, Ala, AIB; (j) Met at position 27 substitutedwith Leu or Nle; (k) Asn at position 29 substituted with a charged aminoacid, optionally, Asp or Glu; and (1) Thr at position 29 substitutedwith Gly or a charged amino acid, optionally, Asp or Glu.

In exemplary aspects, the GIP agonist peptide comprises the amino acidsequence of SEQ ID NO:1 with at least one amino acid modification(optionally, up to 15 amino acid modifications), an extension of 1 to 21amino acids C-terminal to the amino acid at position 29 of the GIPagonist peptide, and an amino acid modification at position 1 whichmodification confers GIP agonist activity. In alternative exemplaryaspects, the GIP agonist peptide does not comprise an amino acidmodification at position 1 which modification confers GIP agonistactivity. In some aspects, the amino acid at position 1 is not a large,aromatic amino acid, e.g., Tyr. In some embodiments, the amino acid atposition 1 is an amino acid comprising an imidazole ring, e.g., His,analogs of His. In certain embodiments, the GIP agonist peptide is notany of the compounds disclosed in U.S. Patent Application No.61/151,349. In certain aspects, the GIP agonist peptide comprises theamino acid sequence of any of SEQ ID NOs: 1057-1069. In some aspects,the GIP agonist peptide comprises a modified amino acid sequence of anyof the glucagon-based sequences of any of SEQ ID NOs: 402-1056 in whichthe referenced amino acid sequence is modified to have (if it does notalready have) (i) a stabilized alpha helix in the C-terminal portion ofthe peptide (e.g., amino acids 12-29), e.g., stabilized via anintramolecular bridge (e.g., a lactam bridge, a salt bridge) asdescribed herein or stabilized via incorporation of one or more alpha,alpha disubstituted amino acids, e.g., AIB, at, for example, positions16, 20, 21, 24 of the peptide; (ii) Leu at position 27, Ala at position28, and Gly at position 29, or conservative amino acid substitutionsthereof; (iii) Ile at position 12, or a conservative amino acidsubstitution thereof, and optionally, (iv) an amino acid modification atposition 1 which confers GIP activity as described herein, e.g., His 1substituted with a large aromatic amino acid, e.g., Tyr. In someaspects, the alpha, alpha disubstituted amino acid is at position 20 andposition 16 is a positive-charged amino acid, e.g., an amino acid ofFormula IV, e.g., Lys.

With regard to the GIP agonist peptides which comprise an extension of1-21 amino acids (e.g., 5-19, 7-15, 9-12 amino acids), the extension ofthe GIP agonist peptide may comprise any amino acid sequence, providedthat the extension is 1 to 21 amino acids. In some aspects, theextension is 7 to 15 amino acids and in other aspects, the extension is9 to 12 amino acids. In some embodiments, the extension comprises (i)the amino acid sequence of SEQ ID NO: 426 or 1074, (ii) an amino acidsequence which has high sequence identity (e.g., at least 80%, 85%, 90%,95%, 98%, 99%) with the amino acid sequence of SEQ ID NO: 426 or 1074,or (iii) the amino acid sequence of (i) or (ii) with one or moreconservative amino acid modifications.

In some embodiments, at least one of the amino acids of the extension isacylated or alkylated. The amino acid comprising the acyl or alkyl groupmay be located at any position of extension of the GIP agonist peptide.In certain embodiments, the acylated or alkylated amino acid of theextension is located at one of positions 37, 38, 39, 40, 41, or 42(according to the numbering of SEQ ID NO: 1) of the GIP agonist peptide.In certain embodiments, the acylated or alkylated amino acid is locatedat position 40 of the GIP agonist peptide.

In exemplary embodiments, the acyl or alkyl group is an acyl or alkylgroup which is non-native to a naturally-occurring amino acid. Forexample, the acyl or alkyl group may be a C4 to C30 (e.g., C12 to C18)fatty acyl group or C4 to C30 (e.g., C12 to C18) alkyl. The acyl oralkyl group may be any of those discussed herein.

In some embodiments, the acyl or alkyl group is attached directly to theamino acid, e.g., via the side chain of the amino acid. In otherembodiments, the acyl or alkyl group is attached to the amino acid via aspacer (e.g., an amino acid, a dipeptide, a tripeptide, a hydrophilicbifunctional spacer, a hydrophobic bifunctional spacer). In certainaspects, the spacer is 3 to 10 atoms in length. In some embodiments, thespacer is an amino acid or dipeptide comprising one or two of6-aminohexanoic acid, Ala, Pro, Leu, beta-Ala, gamma-Glu (e.g.,gamma-Glu-gamma-Glu). In particular aspects, the total length of thespacer is 14 to 28 atoms.

Also, in exemplary embodiments, the amino acid to which the acyl oralkyl group is attached may be any of those described herein, including,for example, an amino acid of Formula I, II, or III. The amino acidwhich is acylated or alkylated may be a Lys, for example. Suitable aminoacids comprising an acyl or alkyl group, as well as suitable acylgroups, alkyl groups, and spacers are described herein. See, forexample, the teachings under the sections entitled Acylation andAlkylation.

In other embodiments, 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 aminoacids) of the extension are positive-charged amino acids, e.g., Arg,amino acids of Formula IV, such as, for example, Lys, D-Lys. As usedherein, the term “positive-charged amino acid” refers to any amino acid,naturally-occurring or non-naturally occurring, comprising a positivecharge on an atom of its side chain at a physiological pH. In certainaspects, the positive-charged amino acids are located at any ofpositions 37, 38, 39, 40, 41, 42, and 43. In specific embodiments, apositive-charged amino acid is located at position 40.

In other embodiments, 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 aminoacids) of the extension are negative-charged amino acids, e.g., Asp,Glu. As used herein, the term “negative-charged amino acid” refers toany amino acid, naturally-occurring or non-naturally occurring,comprising a negative charge on an atom of its side chain at aphysiological pH. In certain aspects, the negative-charged amino acidsare located at any of positions 37, 38, 39, 40, 41, 42, and 43. Inspecific embodiments, a negative-charged amino acid is located atposition 40.

In other instances, the extension is acylated or alkylated as describedherein and comprises 1-6 positive charged amino acids as describedherein.

In yet other embodiments, the GIP agonist peptides which exhibit agonistactivity at the GIP receptor comprises (i) SEQ ID NO: 1 with at leastone amino acid modification, (ii) an extension of 1 to 21 amino acids(e.g., 5 to 18, 7 to 15, 9 to 12 amino acids) C-terminal to the aminoacid at position 29 of the GIP agonist peptide, and (iii) an amino acidcomprising an acyl or alkyl group which is non-native to anaturally-occurring amino acid which is located outside of theC-terminal extension (e.g., at any of positions 1-29). In someembodiments, the GIP agonist peptide comprises an acylated or alkylatedamino acid at position 10. In particular aspects, the acyl or alkylgroup is a C4 to C30 fatty acyl or C4 to C30 alkyl group. In someembodiments, the acyl or alkyl group is attached via a spacer, e.g., anamino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer,hydrophobic bifunctional spacer). In certain aspects, the GIP agonistpeptide comprises an amino acid modification which stabilizes the alphahelix, such as a salt bridge between a Glu at position 16 and a Lys atposition 20, or an alpha, alpha-disubstituted amino acid at any one,two, three, or more of positions 16, 20, 21, and 24. In specificaspects, the GIP agonist peptide additionally comprises amino acidmodifications which confer DPP-IV protease resistance, e.g., DMIA atposition 1, AIB at position 2. GIP agonist peptides comprising furtheramino acid modifications are contemplated herein.

In certain embodiments, the GIP agonist peptides having GIP receptoractivity exhibit at least 0.1% (e.g., at least 0.5%, 1%, 2%, 5%, 10%,15%, or 20%) activity of native GIP at the GIP receptor when the GIPagonist peptide lacks a hydrophilic moiety, e.g., PEG. In someembodiments, the GIP agonist peptides exhibit more than 10%, (e.g., morethan 20%, more than 50%, more than 75%, more than 100%, more than 200%,more than 300%, more than 500%) activity of native GIP at the GIPreceptor. In some embodiments, the GIP agonist peptide exhibitsappreciable agonist activity at one or both of the GLP-1 and glucagonreceptors. In some aspects, the potency and/or selectivity for thesereceptors (GIP receptor and GLP-1 receptor and/or glucagon receptor) arewithin 1000-fold, 750-fold, 500-fold, 250-fold, or 100-fold (higher orlower). For example, the selectivity for the GLP-1 receptor of the GIPagonist peptides having GIP receptor activity can be less than1000-fold, 500-fold, 100-fold, within 50-fold, within 25 fold, within 15fold, within 10 fold) (higher or lower) the selectivity for the GIPreceptor and/or the glucagon receptor.

In accordance with the foregoing, in some embodiments, the GIP agonistpeptide is an analog of native glucagon (SEQ ID NO: 1) and the aminoacid sequence of the peptide is SEQ ID NO: 1 with a stabilized alphahelix (e.g., a lactam bridge or alpha, alpha disubstituted amino acids),Leu-Ala-Gly as positions 27-29, Ile at position 12, an amino acid atposition 1 that confers the peptide with GIP activity or an amino acidwhich reduces GIP activity as described herein. In accordance with theforegoing, in some embodiments, the GIP agonist peptide is an analog ofnative glucagon (SEQ ID NO: 1) and the amino acid sequence of thepeptide is SEQ ID NO: 1 with an amino acid of Formula IV at position 16,and alpha, alpha disubstituted amino acid (e.g., AIB) at position 20,Leu-Ala-Gly as positions 27-29, Ile at position 12, an amino acid atposition 1 that confers the peptide with GIP activity or an amino acidwhich reduces GIP activity as described herein.

In some embodiments, the GIP agonist peptide comprises a modificationthat reduces activity at the GIP receptor at position 1. The GIP agonistpeptide may comprise such an amino acid modification so that theactivity levels at the GIP receptor (e.g., EC50 or potency at the GIPreceptor) are within about 50-fold, about 40-fold, about 30-fold, about20 fold, about 10 fold, or about 5-fold of the IC50 of the glucagonantagonist of the peptide combination. In certain aspects, the aminoacid modification which reduces GIP agonist activity is a substitutionof His at position 1 with a small aliphatic residue, e.g., Ala, Gly. Insome aspects, the amino acid modification which reduces GIP agonistactivity is a deletion of the amino acid at position 1 or a deletion ofthe amino acids at positions 1 and 2. In specific aspects, the GIPagonist peptide is an analog of any of the amino acid sequences listedin Sequence Listing 2 in which Tyr is at position 1, wherein the analogcomprises a small aliphatic residue at position 1, in lieu of the Tyr,or the analog lacks the amino acid at position 1 or at positions 1 and 2of these amino acid sequences.

The following paragraphs which precede the section entitled “Activity ofthe Glucagon antagonist peptide” are provided to further describe theamino acid modifications referenced in the above section entitled“Exemplary embodiments of the GIP agonist peptide” and the sectionentitled “Additional modifications of the Glucagon antagonist peptide.”

Modifications that Affect GIP Activity

Under normal circumstances, native human glucagon does not activate theGIP receptor in the human body. Described herein are modifications ofthe native human glucagon amino acid sequence which alter this hormone,such that it exhibits appreciable activity at the GIP receptor.

In specific aspects, the GIP agonist peptide of the present disclosureswhich exhibits enhanced activity at the GIP receptor (as compared tonative glucagon) comprises an amino acid modification at position 1. Forexample, the His at position 1 of native glucagon is substituted with alarge, aromatic amino acid, optionally Tyr, Phe, Trp, amino-Phe,nitro-Phe, chloro-Phe, sulfo-Phe, 4-pyridyl-Ala, methyl-Tyr, or 3-aminoTyr in the GIP agonist peptide.

In alternative embodiments, the GIP agonist peptide comprises amodification that reduces activity at the GIP receptor at position 1.The GIP agonist peptide may comprise such an amino acid modification sothat the activity levels at the GIP receptor (e.g., EC50 or potency atthe GIP receptor) are within about 50-fold, about 40-fold, about30-fold, about 20 fold, about 10 fold, or about 5-fold of the IC50 ofthe glucagon antagonist of the peptide combination. In certain aspects,the amino acid modification which reduces GIP agonist activity is asubstitution of His at position 1 with a small aliphatic residue, e.g.,Ala, Gly. In some aspects, the amino acid modification which reduces GIPagonist activity is a deletion of the amino acid at position 1 or adeletion of the amino acids at positions 1 and 2.

In some embodiments, the GIP agonist peptide which exhibits enhancedactivity at the GIP receptor (as compared to native glucagon) comprisesan amino acid modification at one or all of positions 27, 28, and 29. Insome embodiments, the GIP agonist peptide comprises the amino acidsequence of native glucagon in which (i) the Met at position 27 of thenative glucagon amino acid sequence is substituted with a largealiphatic amino acid, optionally Leu, (ii) the Asn at position 28 of thenative glucagon amino acid sequence is substituted with a smallaliphatic amino acid, optionally Ala, (iii) the Thr at position 29 ofthe native glucagon amino acid sequence is substituted with a smallaliphatic amino acid, optionally Gly, or any combination of (i), (ii),and (iii). Substitution with LAG at positions 27-29 provides increasedGIP activity relative to the MNT sequence of native human glucagon atthose positions.

In some embodiments, the GIP agonist peptide which exhibits enhancedactivity at the GIP receptor (as compared to native glucagon) comprisesan amino acid modification at position 12. For example, in some aspects,the amino acid at position 12 of the native glucagon amino acid sequenceis substituted with a large, aliphatic, nonpolar amino acid, optionallyIle.

In certain aspects, the GIP agonist peptide which exhibits enhancedactivity at the GIP receptor (as compared to native glucagon) comprisesan amino acid modification at positions 17 and/or 18. For example,position 17 is substituted with a polar residue, optionally Gln, andposition 18 is substituted with a small aliphatic amino acid, optionallyAla. A substitution with QA at positions 17 and 18 provides increasedGIP activity relative to the native RR sequence at those positions.

In some embodiments, the GIP agonist peptide which exhibits increasedactivity at the GIP receptor (as compared to native glucagon) comprisesan intramolecular bridge between the side chains of two amino acidslocated at any of positions from 12 to 29. In some aspects, theintramolecular bridge is formed between two amino acids that are notpresent in the native amino acid sequence of human glucagon.Accordingly, the GIP agonist peptide in some aspects comprises aminoacid modifications, e.g., amino acid substitutions of the native humanglucagon sequence, that permit bridge formation. For example, in someaspects, an intramolecular bridge is formed by a covalent bond betweenthe side chains of two amino acids at positions i and i+4 or betweenpositions j and j+3, or between positions k and k+7. In exemplaryembodiments, the bridge is between positions 12 and 16, 16 and 20, and24, 24 and 28, or 17 and 20. In other embodiments, non-covalentinteractions such as salt bridges can be formed between positively andnegatively charged amino acids at these positions. Intramolecularbridges within the C-terminal region of a glucagon-based peptide arefurther described herein. See “Stabilization of the Alpha HelixStructure”

In some embodiments, stabilization of the alpha helix structure in theC-terminal portion of the glucagon peptide (around amino acids 12-29) isachieved through purposeful introduction of one or moreα,α-disubstituted amino acids at positions that retain the desiredactivity. In some embodiments, one, two, three, four or more ofpositions 16, 17, 18, 19, 20, 21, 24 or 29 of a glucagon peptide oranalog thereof is substituted with an α,α-disubstituted amino acid. Forexample, substitution of position 16 of a glucagon peptide or analogthereof with amino iso-butyric acid (AIB) provides a stabilized alphahelix in the absence of a salt bridge or lactam. Such peptides areconsidered herein as a peptide lacking an intramolecular bridge. Inspecific aspects, stabilization of the alpha-helix is accomplished byintroducing one or more α,α-disubstituted amino acids withoutintroduction of a covalent intramolecular bridge, e.g., a lactam bridge,a disulfide bridge. Such peptides are considered herein as a peptidelacking a covalent intramolecular bridge. In some embodiments, one, two,three or more of positions 16, 20, 21 or 24 are substituted with AIB.Further discussion of this type of amino acid modification is providedherein. See “Stabilization of the Alpha Helix Structure”

In some embodiments, the GIP agonist peptide comprises an extension of1-21 amino acids C-terminal to the amino acid at position 29. Theextension in some aspects comprises the amino acid sequence of SEQ IDNO: 3 or 4, for instance. In some aspects in which the extensioncomprises SEQ ID NO: 4, the Xaa is a small aliphatic residue, e.g., Gly.In some aspects, the extension comprises 1-6 charged amino acids. Insome embodiments, the 1-6 amino acids are negative-charged amino acids,e.g., Asp, Glu. In some embodiments, the 1-6 amino acids arepositive-charged amino acids, e.g., Arg, an amino acid of Formula IV(e.g., Dab, Orn, Lys, d-Lys, homoLys). The charged amino acid may belocated at any of positions 37, 38, 39, 40, 41, 42, and 43. In someaspects, the charged amino acid is located at position 40. In furtheraspects, the charged amino acid is modified with an acyl or alkyl groupas described herein in “Acylation and alkylation.” In some aspects, theextension does not comprise a Lys at position 40.

Any of the modifications described above which provide an enhancement inGIP receptor activity can be applied individually or in combination.Combinations of the modifications that increase GIP receptor activitygenerally provide higher GIP activity than any of such modificationstaken alone.

Modifications that Affect Glucagon Activity

In some aspects of the present disclosures, the GIP agonist peptidewhich is an analog of native human glucagon comprises an amino acidmodification that selectively reduces glucagon receptor activity. Inspecific embodiments, the modification that reduces glucagon receptoractivity is a modification of the amino acid at position 3, e.g.substitution of the naturally occurring glutamine at position 3, with anacidic, basic, or a hydrophobic amino acid. In exemplary embodiments,the GIP agonist peptide comprises the native human glucagon amino acidsequence in which the amino acid at position 3 is substituted withglutamic acid, ornithine, or norleucine. Such modifications have beenfound to substantially reduce or destroy glucagon receptor activity.Without being bound to any particular theory, such amino acidsubstitutions at position 3 dominate any other amino acid modificationwhich enhances glucagon receptor activity, such that the net result isreduced or destroyed activity at the glucagon receptor.

Modifications that Affect GLP-1 Activity

Under normal circumstances, native human glucagon does not activate theGLP-1 receptor in the human body. Described herein are modifications ofthe native human glucagon amino acid sequence which alter this hormone,such that it exhibits appreciable activity at the GLP-1 receptor.

Accordingly, in some embodiments, the GIP agonist peptide of the presentdisclosures exhibits enhanced activity at both the GIP receptor andGLP-1 receptor (as compared to the activity of native glucagon at thesereceptors). In this regard, the GIP agonist peptide may be considered asa GIP/GLP-1 co-agonist peptide. The GIP agonist peptide which exhibitsenhanced activity at the GLP-1 receptor in some embodiments comprises acharge-neutral group, such as an amide or ester, in place of the alphacarboxylic acid of the C-terminal amino acid. Thus, in some aspects, theGIP agonist peptide comprises C-terminal amidation or comprises aC-terminal amide in place of the alpha carboxylate of the C-terminalresidue.

In some aspects, the GIP agonist peptide which exhibits enhancedactivity at the GLP-1 receptor comprises a stabilized alpha-helixstructure in the C-terminal portion of glucagon (around amino acids12-29), e.g., through formation of an intramolecular bridge between theside chains of two amino acids, or substitution and/or insertion ofamino acids around positions 12-29 with an alpha helix-stabilizing aminoacid (e.g., an α,α-disubstituted amino acid), as further describedherein. In exemplary embodiments, the side chains of the amino acidpairs 12 and 16, 13 and 17, 16 and 20, 17 and 21, 20 and 24 or 24 and 28(amino acid pairs in which i=12, 16, 20, or 24) are linked to oneanother and thus stabilize the glucagon alpha helix. In someembodiments, the bridge or linker is about 8 (or about 7-9) atoms inlength, particularly when the bridge is between positions i and i+4. Insome embodiments, the bridge or linker is about 6 (or about 5-7) atomsin length, particularly when the bridge is between positions j and j+3.

In some embodiments, intramolecular bridges are formed by (a)substituting the naturally occurring serine at position 16 with glutamicacid or with another negatively charged amino acid having a side chainwith a length of 4 atoms, or alternatively with any one of glutamine,homoglutamic acid, or homocysteic acid, or a charged amino acid having aside chain containing at least one heteroatom, (e.g. N, O, S, P) andwith a side chain length of about 4 (or 3-5) atoms, and (b) substitutingthe naturally occurring glutamine at position 20 with anotherhydrophilic amino acid having a side chain that is either charged or hasan ability to hydrogen-bond, and is at least about 5 (or about 4-6)atoms in length, for example, lysine, citrulline, arginine, orornithine. The side chains of such amino acids at positions 16 and 20can form a salt bridge or can be covalently linked. In some embodimentsthe two amino acids are bound to one another to form a lactam ring.

In some embodiments, stabilization of the alpha helix structure in theC-terminal portion of the GIP agonist peptide is achieved through theformation of an intramolecular bridge other than a lactam bridge. Forexample, suitable covalent bonding methods include any one or more ofolefin metathesis, lanthionine-based cyclization, disulfide bridge ormodified sulfur-containing bridge formation, the use ofα,ω-diaminoalkane tethers, the formation of metal-atom bridges, andother means of peptide cyclization are used to stabilize the alphahelix.

In yet other embodiments, one or more α,α-disubstituted amino acids areinserted or substituted into this C-terminal portion (amino acids 12-29)at positions that retain the desired activity. For example, one, two,three or all of positions 16, 20, 21 or 24 are substituted with anα,α-disubstituted amino acid, e.g., AIB.

In some aspects, the GIP agonist peptide which exhibits increasedactivity at the GLP-1 receptor comprises an amino acid modification atposition 20 as described herein.

In some embodiments, the GIP agonist peptide which exhibits increasedactivity at the GLP-1 receptor comprises GPSSGAPPPS (SEQ ID NO: 3) orXGPSSGAPPPS (SEQ ID NO: 4) at the C-terminus. GLP-1 activity in suchanalogs can be further increased by modifying the amino acid at position18, 28 or 29, or at position 18 and 29, as described herein.

In some embodiments, the GIP agonist peptide which exhibits increasedactivity at the GLP-1 receptor comprises a large, aromatic amino acidresidue, optionally Trp, at position 10.

In some embodiments in which the GIP agonist peptide exhibits enhancedactivity at the GLP-1 receptor, the GIP agonist peptide comprises analanine at position 18 instead of an arginine which is native to thehuman glucagon amino acid sequence.

Any of the modifications described above in reference to a GIP agonistpeptide which exhibit increased GLP-1 receptor activity can be appliedindividually or in combination. Combinations of the modifications thatincrease GLP-1 receptor activity generally provide higher GLP-1 activitythan any of such modifications taken alone. For example, the presentdisclosures provides GIP agonist peptides that comprise modifications atposition 16, at position 20, and at the C-terminal carboxylic acidgroup, optionally with a covalent bond between the amino acids atpositions 16 and 20; GIP agonist peptides that comprise modifications atposition 16 and at the C-terminal carboxylic acid group; GIP agonistpeptides that comprise modifications at positions 16 and 20, optionallywith a covalent bond between the amino acids at positions 16 and 20; andGIP agonist peptides that comprise modifications at position 20 and atthe C-terminal carboxylic acid group.

In some embodiments in which GLP-1 activity is not desired, GLP-1activity may be reduced by specific amino acid modifications. In thisregard, in some embodiments, the GIP agonist peptide comprises (i) aC-terminal alpha carboxylate group, (ii) a substitution of the Thr atposition 7 with an amino acid lacking a hydroxyl group, e.g., Abu orIle, (iii) a deletion of the amino acid(s) C-terminal to the amino acidat position 27 or 28 (e.g., deletion of the amino acid at position 28,deletion of the amino acid at positions 28 and 29) to yield a peptide 27or 28 amino acids in length, or (iv) a combination thereof. In someaspects, an amino acid substitution at position 7 which reduces, if notdestroys, GLP-1 receptor activity dominates any other amino acidmodification which is described herein as one which enhances GLP-1receptor activity, such that the net effect of the modifications wouldbe reduced or destroyed activity at the GLP-1 receptor.

Modifications that Affect Activity at Each of the GIP, GLP-1, andGlucagon Receptors

In some embodiments, the GIP agonist peptide comprises (i) an amino acidsubstitution of Ser at position 16 with an amino acid of Formula IV:

wherein n is 1 to 16, or 1 to 10, or 1 to 7, or 1 to 6, or 2 to 6, or 2or 3 or 4 or 5, each of R₁ and R₂ is independently selected from thegroup consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group, and (ii) anamino acid substitution of the Gln at position 20 with an alpha,alpha-disubstituted amino acid, e.g., AIB. In some embodiments, theamino acid at position 16 is Orn, Dab, Lys, or homoLys, and the aminoacid at position 20 is AIB. In specific embodiments, the amino acid atposition 16 is Lys and the amino acid at position 20 is AIB.

The activity at each of the glucagon receptor, GLP-1 receptor, andglucagon receptor of the GIP agonist peptide comprising an amino acid ofFormula IV at position 16 and an alpha, alpha di-substituted amino acidat position 20 can be further enhanced by extending the length of thepeptide, e.g. by fusion to a C-terminal extension peptide, e.g. of about1-21, about 9 to 21, about 6-18, about 9-12, or about 10 or 11 aminoacids in length. In some embodiments, the C-terminus of the GIP agonistpeptide is extended by fusion to GPSSGAPPPS (SEQ ID NO: 3) orXGPSSGAPPPS (SEQ ID NO: 4), wherein X is Gly or a small, aliphatic ornon-polar or slightly polar amino acid. In alternative embodiments, theC-terminus of the GIP agonist peptide is extended by fusion toGPSSGAPPPS (SEQ ID NO: 3) and 1-11 amino acids are fused to theC-terminus of GPSSGAPPPS (SEQ ID NO: 3). For example, the C-terminalextension of the analog can comprise GPSSGAPPPS (SEQ ID NO: 3) followedby 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 additional amino acids at theC-terminus of SEQ ID NO: 3. The 1-11 additional amino acids can be, forexample, a small aliphatic amino acid, such as Ala. In this regard, theGIP agonist peptide in some embodiments comprises a C-terminal extensioncomprising, for example, the amino acid sequence of GPSSGAPPPSA_(m),wherein m is 1 to 11.

Enhancement of activity at each of the glucagon, GLP-1, and GIPreceptors of the GIP agonist peptide, including one comprising an aminoacid of Formula IV at position 16 and an alpha, alpha disubstitutedamino acid at position 20, can furthermore be achieved upon acylation oralkylation of an amino acid located within a C-terminal extension or atthe C-terminal amino acid (e.g., an amino acid which is added to theC-terminus of the C-terminal extension). The acylation or alkylation canbe of an amino acid located, for example, at any of positions 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,and 50 of the C-terminally extended GIP agonist peptide. In someembodiments, the amino acid which is acylated or alkylated is located atposition 37, 38, 39, 40, 41, 42, or 43 of the C-terminally extended GIPagonist peptide. In some embodiments, the acylated or alkylated aminoacid is an amino acid of Formula I, II, or III, e.g., Lys, which isattached to an acyl or alkyl group, e.g. C10-C22. In certainembodiments, the Lys is located C-terminal to a C-terminal extensionconsisting of SEQ ID NO: 3, such that the Lys, Dab, Orn, or homoLys islocated at position 40 of the analog. Optionally, C-terminally extendedpeptides are also pegylated, e.g. at any of the positions describedherein as suitable for pegylation (e.g., position 24).

Enhancement of the activity at each of the glucagon, GLP-1, and GIPreceptors of a GIP-active, GIP agonist peptide can moreover be achievedby acylation or alkylation of an amino acid via a spacer (e.g., an aminoacid, dipeptide, tripeptide, hydrophilic bifunctional spacer,hydrophobic bifunctional spacer). In some embodiments, the GIP-active,GIP agonist peptide comprises an acyl or alkyl group via a spacer, whichspacer is attached to the side chain of the amino acid at position 10 ofthe analog. In other embodiments, the GIP agonist peptide comprises aC-terminal extension of 1 to 21 amino acids (e.g., an extensioncomprising the amino acid sequence of SEQ ID NO: 3 or 4) C-terminal tothe amino acid at position 29 and the spacer, which is covalentlyattached to an acyl or alkyl group, is attached to an amino acid of theextension at a position corresponding to one of positions 37-43 relativeto SEQ ID NO: 1. In specific embodiments, the spacer is attached to theamino acid at position 40 relative to SEQ ID NO: 1. In certainembodiments, the spacer is 3 to 10 atoms in length. In specific aspects,the total length of the spacer and acyl or alkyl group is about 14 toabout 28 atoms in length. For example, the spacer can be an amino acid,including, but not limited to, any of those described herein. Also, forexample, the spacer may be a dipeptide or tripeptide comprising aminoacids described herein, e.g., a dipeptide or tripeptide spacercomprising acidic amino acids. The spacer in specific aspects is one ofthe following dipeptides: Ala-Ala, βAla-βAla, or γGlu-γGlu. Additionalsuitable spacers for purposes of increasing activity at one or more ofthe glucagon, GLP-1, and GIP receptors are further described herein.

In particular aspects of the present disclosures, the GIP agonistpeptide comprises any of the modifications above which achieve enhancedactivity at each of the GIP, GLP-1, and glucagon receptors, in additionto an amino acid modification which reduces glucagon activity, e.g., Gluat position 3. Without being bound to any particular theory, an aminoacid substitution at position 3 which reduces, if not destroys, glucagonreceptor activity dominates any other amino acid modification whichenhances glucagon receptor activity, such that the net results would bereduced or destroyed activity at the glucagon receptor. Accordingly, inexemplary embodiments, the GIP agonist peptide comprises an amino acidof Formula IV at position 16, an alpha, alpha-disubstituted amino acidat position 20, a C-terminal extension in accordance with the aboveteachings comprising an acylated Lys residue at position 40, and a Gluat position 3. Such peptides exhibits little to no glucagon receptoractivity.

Stabilization of the Alpha Helix Structure

Stabilization of the alpha-helix structure in the C-terminal portion ofthe GIP agonist peptide (around amino acids 12-29) provides enhancedGLP-1 and/or GIP activity and restores glucagon activity which has beenreduced by amino acid modifications at positions 1 and/or 2. The alphahelix structure can be stabilized by, e.g., formation of a covalent ornon-covalent intramolecular bridge, or substitution and/or insertion ofamino acids around positions 12-29 with an alpha helix-stabilizing aminoacid (e.g., an α,α-disubstituted amino acid).

In some embodiments, an intramolecular bridge is formed between twoamino acid side chains to stabilize the three dimensional structure ofthe carboxy terminal portion (e.g., amino acids 12-29) of the GIPagonist peptide. The two amino acid side chains can be linked to oneanother through non-covalent bonds, e.g., hydrogen-bonding, ionicinteractions, such as the formation of salt bridges, or by covalentbonds. When the two amino acid side chains are linked to one anotherthrough one or more covalent bonds, the peptide may be considered hereinas comprising a covalent intramolecular bridge. When the two amino acidside chains are linked to one another through non-covalent bonds, e.g.,hydrogen bonds, ionic interactions, the peptide may be considered hereinas comprising a non-covalent intramolecular bridge.

In some embodiments, the intramolecular bridge is formed between twoamino acids that are 3 amino acids apart, e.g., amino acids at positionsi and i+4, wherein i is any integer between 12 and 25 (e.g., 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25). More particularly, theside chains of the amino acid pairs 12 and 16, 16 and 20, 20 and 24 or24 and 28 (amino acid pairs in which i=12, 16, 20, or 24) are linked toone another and thus stabilize the glucagon alpha helix. Alternatively,i can be 17.

In some specific embodiments, wherein the amino acids at positions i andi+4 are joined by an intramolecular bridge, the size of the linker isabout 8 atoms, or about 7-9 atoms.

In other embodiments, the intramolecular bridge is formed between twoamino acids that are two amino acids apart, e.g., amino acids atpositions j and j+3, wherein j is any integer between 12 and 26 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26). In somespecific embodiments, j is 17.

In some specific embodiments, wherein amino acids at positions j and j+3are joined by an intramolecular bridge, the size of the linker is about6 atoms, or about 5 to 7 atoms.

In yet other embodiments, the intramolecular bridge is formed betweentwo amino acids that are 6 amino acids apart, e.g., amino acids atpositions k and k+7, wherein k is any integer between 12 and 22 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22). In some specificembodiments, k is 12, 13, or 17. In an exemplary embodiment, k is 17.

Examples of amino acid pairings that are capable of covalently bondingto form a six-atom linking bridge include Orn and Asp, Glu and an aminoacid of Formula I, wherein n is 2, and homoglutamic acid and an aminoacid of Formula I, wherein n is 1, wherein Formula I is:

Examples of amino acid pairing that are capable of covalently bonding toform a seven-atom linking bridge include Orn-Glu (lactam ring); Lys-Asp(lactam); or Homoser-Homoglu (lactone). Examples of amino acid pairingsthat may form an eight-atom linker include Lys-Glu (lactam); Homolys-Asp(lactam); Orn-Homoglu (lactam); 4-aminoPhe-Asp (lactam); or Tyr-Asp(lactone). Examples of amino acid pairings that may form a nine-atomlinker include Homolys-Glu (lactam); Lys-Homoglu (lactam);4-aminoPhe-Glu (lactam); or Tyr-Glu (lactone). Any of the side chains onthese amino acids may additionally be substituted with additionalchemical groups, so long as the three-dimensional structure of thealpha-helix is not disrupted. One of ordinary skill in the art canenvision alternative pairings or alternative amino acid analogs,including chemically modified derivatives, that would create astabilizing structure of similar size and desired effect. For example, ahomocysteine-homocysteine disulfide bridge is 6 atoms in length and maybe further modified to provide the desired effect. Even without covalentlinkage, the amino acid pairings described above or similar pairingsthat one of ordinary skill in the art can envision may also provideadded stability to the alpha-helix through non-covalent bonds, forexample, through formation of salt bridges or hydrogen-bondinginteractions.

The size of a lactam ring can vary depending on the length of the aminoacid side chains, and in one embodiment the lactam is formed by linkingthe side chains of a lysine amino acid to a glutamic acid side chain.Further exemplary embodiments include the following pairings, optionallywith a lactam bridge: Glu at position 12 with Lys at position 16; nativeLys at position 12 with Glu at position 16; Glu at position 16 with Lysat position 20; Lys at position 16 with Glu at position 20; Glu atposition 20 with Lys at position 24; Lys at position 20 with Glu atposition 24; Glu at position 24 with Lys at position 28; Lys at position24 with Glu at position 28. Alternatively, the order of the amide bondin the lactam ring can be reversed (e.g., a lactam ring can be formedbetween the side chains of a Lys12 and a Glu16 or alternatively betweena Glu 12 and a Lys16).

Intramolecular bridges other than a lactam bridge can be used tostabilize the alpha helix of the GIP agonist peptides. In oneembodiment, the intramolecular bridge is a hydrophobic bridge. In thisinstance, the intramolecular bridge optionally is between the sidechains of two amino acids that are part of the hydrophobic face of thealpha helix of the GIP agonist peptide. For example, one of the aminoacids joined by the hydrophobic bridge can be the amino acid at position10, 14, and 18.

In one specific aspect, olefin metathesis is used to cross-link one ortwo turns of the alpha helix of the GIP agonist peptide using anall-hydrocarbon cross-linking system. The GIP agonist peptide in thisinstance can comprise α-methylated amino acids bearing olefinic sidechains of varying length and configured with either R or Sstereochemistry at the and i+4 or i+7 positions. For example, theolefinic side can comprise (CH₂)n, wherein n is any integer between 1 to6. In one embodiment, n is 3 for a cross-link length of 8 atoms.Suitable methods of forming such intramolecular bridges are described inthe art. See, for example, Schafineister et al., J. Am. Chem. Soc. 122:5891-5892 (2000) and Walensky et al., Science 305: 1466-1470 (2004).Alternatively, the GIP agonist peptide can comprise O-allyl Ser residueslocated on adjacent helical turns, which are bridged together viaruthenium-catalyzed ring closing metathesis. Such procedures ofcross-linking are described in, for example, Blackwell et al., Angew,Chem., Int. Ed. 37: 3281-3284 (1998).

In another specific aspect, use of the unnatural thio-dialanine aminoacid, lanthionine, which has been widely adopted as a peptidomimetic ofcystine, is used to cross-link one turn of the alpha helix. Suitablemethods of lanthionine-based cyclization are known in the art. See, forinstance, Matteucci et al., Tetrahedron Letters 45: 1399-1401 (2004);Mayer et al., J. Peptide Res. 51: 432-436 (1998); Polinsky et al., J.Med. Chem. 35: 4185-4194 (1992); Osapay et al., J. Med. Chem. 40:2241-2251 (1997); Fukase et al., Bull. Chem. Soc. Jpn. 65: 2227-2240(1992); Harpp et al., J. Org. Chem. 36: 73-80 (1971); Goodman and Shao,Pure Appl. Chem. 68: 1303-1308 (1996); and Osapay and Goodman, J. Chem.Soc. Chem. Commun. 1599-1600 (1993).

In some embodiments, α,ω-diaminoalkane tethers, e.g., 1,4-diaminopropaneand 1,5-diaminopentane) between two Glu residues at positions i and i+7are used to stabilize the alpha helix of the GIP agonist peptide. Suchtethers lead to the formation of a bridge 9-atoms or more in length,depending on the length of the diaminoalkane tether. Suitable methods ofproducing peptides cross-linked with such tethers are described in theart. See, for example, Phelan et al., J. Am. Chem. Soc. 119: 455-460(1997).

In yet another embodiment of the present disclosures, a disulfide bridgeis used to cross-link one or two turns of the alpha helix of the GIPagonist peptide. Alternatively, a modified disulfide bridge in which oneor both sulfur atoms are replaced by a methylene group resulting in anisosteric macrocyclization is used to stabilize the alpha helix of theGIP agonist peptide. Suitable methods of modifying peptides withdisulfide bridges or sulfur-based cyclization are described in, forexample, Jackson et al., J. Am. Chem. Soc. 113: 9391-9392 (1991) andRudinger and Jost, Experientia 20: 570-571 (1964).

In yet another embodiment, the alpha helix of the GIP agonist peptide isstabilized via the binding of metal atom by two His residues or a Hisand Cys pair positioned at i and i+4. The metal atom can be, forexample, Ru(III), Cu(II), Zn(II), or Cd(II). Such methods of metalbinding-based alpha helix stabilization are known in the art. See, forexample, Andrews and Tabor, Tetrahedron 55: 11711-11743 (1999); Ghadiriet al., J. Am. Chem. Soc. 112: 1630-1632 (1990); and Ghadiri et al., J.Am. Chem. Soc. 119: 9063-9064 (1997).

The alpha helix of the GIP agonist peptide can alternatively bestabilized through other means of peptide cyclizing, which means arereviewed in Davies, J. Peptide. Sci. 9: 471-501 (2003). The alpha helixcan be stabilized via the formation of an amide bridge, thioetherbridge, thioester bridge, urea bridge, carbamate bridge, sulfonamidebridge, and the like. For example, a thioester bridge can be formedbetween the C-terminus and the side chain of a Cys residue.Alternatively, a thioester can be formed via side chains of amino acidshaving a thiol (Cys) and a carboxylic acid (e.g., Asp, Glu). In anothermethod, a cross-linking agent, such as a dicarboxylic acid, e.g. subericacid (octanedioic acid), etc. can introduce a link between twofunctional groups of an amino acid side chain, such as a free amino,hydroxyl, thiol group, and combinations thereof.

In accordance with one embodiment, the alpha helix of the GIP agonistpeptide is stabilized through the incorporation of hydrophobic aminoacids at positions i and i+4. For instance, i can be Tyr and i+4 can beeither Val or Leu; i can be Phe and i+4 can be Cys or Met; I can be Cysand i+4 can be Met; or i can be Phe and i+4 can be Ile. It should beunderstood that, for purposes herein, the above amino acid pairings canbe reversed, such that the indicated amino acid at position i couldalternatively be located at i+4, while the i+4 amino acid can be locatedat the i position.

In accordance with other embodiments of the present disclosures, thealpha helix is stabilized through incorporation (either by amino acidsubstitution or insertion) of one or more alpha helix-stabilizing aminoacids at the C-terminal portion of the GIP agonist peptide (around aminoacids 12-29). In a specific embodiment, the alpha helix-stabilizingamino acid is an α,α-disubstituted amino acid, including, but notlimited to any of amino iso-butyric acid (AIB), an amino aciddisubstituted with the same or a different group selected from methyl,ethyl, propyl, and n-butyl, or with a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). In some embodiments, one, two,three, four or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 of theGIP agonist peptide is substituted with an α,α-disubstituted amino acid.In a specific embodiment, one, two, three or all of positions 16, 20,21, and 24 are substituted with an α,α-disubstituted amino acid, e.g.,AIB, and the GIP agonist peptide optionally lacks anypurposefully-introduced intramolecular bridges. For example, the GIPagonist peptide can comprise a substitution of position 16 or 20 withAIB in the absence of an intramolecular bridge, e.g., a non-covalentintramolecular bridge (e.g., a salt bridge) or a covalent intramolecularbridge (e.g., a lactam). Such peptides lacking an intramolecular bridgeare advantageously easy to prepare.

In accordance with some embodiments, the GIP agonist peptide lacking anintramolecular bridge comprises one or more substitutions within aminoacid positions 12-29 with an α,α-disubstituted amino acid and an acyl oralkyl group covalently attached to the side chain of an amino acid ofthe GIP agonist peptide, e.g., the amino acid at positions 10 or 40 ofthe GIP agonist peptide. In specific embodiments, the acyl or alkylgroup is non-native to a naturally occurring amino acid. In certainaspects, the acyl or alkyl group is non-native to the amino acid atposition 10. Such acylated or alkylated GIP agonist peptides lacking anintramolecular bridge exhibit enhanced activity at the GLP-1 andglucagon receptors as compared to the non-acylated counterpart peptides.Further enhancement in activity at the GLP-1 and glucagon receptors canbe achieved by the acylated GIP agonist peptides lacking anintramolecular bridge by incorporating a spacer between the acyl oralkyl group and the side chain of the amino acid at positions 10 or 40of the peptide. Acylation and alkylation, with or without incorporatingspacers, are further described herein.

In specific embodiments, the acylated or alkylated GIP agonist peptide,or analog thereof, further comprises a modification which selectivelyreduces activity at the GLP-1 receptor. For example, the acylated oralkylated GIP agonist peptide, or analog thereof, comprises one or acombination of: a C-terminal alpha carboxylate, a deletion of the aminoacids C-terminal to the amino acid at position 27 or 28 (e.g., deletionof the amino acid at position 29, deletion of the amino acids atpositions 28 and 29), a substitution of the Thr at position 7 with alarge, aliphatic, non-polar amino acid, e.g., Ile. In some aspects, theGIP agonist peptide of the present disclosures comprises an amino acidmodification which selectively reduces glucagon receptor activity. Suchmodifications are described further herein.

In some embodiments, position 16 or position 20 is substituted with anα,α-disubstituted amino acid, e.g., AIB. In some embodiments, position20 is substituted with an α,α-disubstituted amino acid, e.g., AIB. Incertain embodiments, position 20 is substituted with anα,α-disubstituted amino acid, e.g., AIB, and position 16 is substitutedwith an amino acid of Formula IV

wherein n is 1 to 16, or 1 to 10, or 1 to 7, or 1 to 6, or 2 to 6, or 2or 3 or 4 or 5, each of R₁ and R₂ is independently selected from thegroup consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group. In particularembodiments, the amino acid of Formula IV is 2,3 diamino propionic acid(DAP), 2,4-diaminobutyric acid (DAB), Orn, Lys or homoLys. Thecombination of an amino acid of Formula IV at position 16 and an alpha,alpha disubstituted amino acid advantageously provides improved activityat each of the glucagon, GLP-1, and GIP receptors. In some aspects, thispeptide further comprises an amino acid modification which selectivelyreduces activity at the glucagon receptor, e.g., a substitution of theGln at position 3 with Glu.

Acylation and Alkylation

In accordance with some embodiments, the GIP agonist peptide of thepresent disclosures are modified to comprise an acyl group or an alkylgroup, e.g., an acyl or alkyl group which is non-native to anaturally-occurring amino acid. Acylation or alkylation can increase thehalf-life of the GIP agonist peptides in circulation. Acylation oralkylation can advantageously delay the onset of action and/or extendthe duration of action at the glucagon and/or GLP-1 receptors and/orimprove resistance to proteases such as DPP-IV and/or improvesolubility. Activity at the glucagon and/or GLP-1 and/or GIP receptorsof the GIP agonist peptide may be maintained after acylation. In someembodiments, the potency of the acylated GIP agonist peptides iscomparable to the unacylated versions of the GIP agonist peptides. Inalternative embodiments, the potency of the acylated GIP agonistpeptides is increased as compared to that of the unacylated version ofthe GIP agonist peptides.

In some embodiments, the GIP agonist peptide is modified to comprise anacyl group or alkyl group covalently linked to the amino acid atposition 10 of the GIP agonist peptide. The GIP agonist peptide mayfurther comprise a spacer between the amino acid at position 10 of theGIP agonist peptide and the acyl group or alkyl group. In someembodiments, the acyl group is a fatty acid or bile acid, or saltthereof, e.g. a C4 to C30 fatty acid, a C8 to C24 fatty acid, cholicacid, a C4 to C30 alkyl, a C8 to C24 alkyl, or an alkyl comprising asteroid moiety of a bile acid. The spacer is any moiety with suitablereactive groups for attaching acyl or alkyl groups. In exemplaryembodiments, the spacer comprises an amino acid, a dipeptide, atripeptide, a hydrophilic bifunctional, or a hydrophobic bifunctionalspacer. In some embodiments, the spacer is selected from the groupconsisting of: Trp, Glu, Asp, Cys and a spacer comprisingNH₂(CH₂CH₂O)n(CH₂)mCOOH, wherein m is any integer from 1 to 6 and n isany integer from 2 to 12. Such acylated or alkylated GIP agonistpeptides may also further comprise a hydrophilic moiety, optionally apolyethylene glycol. Any of the foregoing GIP agonist peptides maycomprise two acyl groups or two alkyl groups, or a combination thereof.

Acylation can be carried out at any position within the GIP agonistpeptide, including any of positions 1-29, a position within a C-terminalextension, or the N- or C-terminal amino acid, provided that GIPactivity (and optionally GLP-1 and/or glucagon activity) is retained, ifnot enhanced. Acylation may occur, for example, at any amino acid whichis added to the amino acid sequence (SEQ ID NO: 1), e.g., at the N- orC-terminus. Nonlimiting examples include positions 1, 5, 10, 11, 12, 13,14, 16, 17, 18, 19, 20, 21, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of the GIPagonist peptide. The acyl group can be covalently linked directly to anamino acid of the GIP agonist peptide, or indirectly to an amino acid ofthe GIP agonist peptide via a spacer, wherein the spacer is positionedbetween the amino acid of the GIP agonist peptide and the acyl group.GIP agonist peptides may be acylated at the same amino acid positionwhere a hydrophilic moiety is linked, or at a different amino acidposition. Nonlimiting examples include acylation at position 10 orposition 40 and pegylation at one or more positions in the C-terminalportion of the GIP agonist peptide, e.g., position 24, 28 or 29, withina C-terminal extension, or at the C-terminus (e.g., through adding aC-terminal Cys).

In some embodiments, the GIP agonist peptide is modified to comprise anextension of about 1 to about 21 amino acids C-terminal to the GIPagonist peptide of SEQ ID NO: 1 or an analog thereof and at least one ofthe amino acids of the extension is acylated or alkylated. For example,the modified GIP agonist peptide may comprise an extension of about 1 toabout 21 amino acids C-terminal to the amino acid at position 29 of theGIP agonist peptide of SEQ ID NO: 1 or analog thereof. Alternatively, ifthe GIP agonist peptide or analog thereof is truncated by one or twoamino acids, the extension of about 1 to about 21 amino acids may beC-terminal to the amino acid at position 27 or 28 of the GIP agonistpeptide or analog thereof. Accordingly, the acylated or alkylated aminoacid within the C-terminal extension can be, for example, any of theamino acids at position 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of the C-terminallyextended GIP agonist peptide. The C-terminal extension in someembodiments comprises the amino acid sequence of SEQ ID NO: 3 or 4. Insome embodiments, the GIP agonist peptide comprises a C-terminalextension comprising the amino acid sequence of SEQ ID NO: 3 and 1 to 11additional amino acids at the C-terminus of SEQ ID NO: 3, whichadditional amino acid(s) is/are acylated or alkylated, as describedherein. In specific embodiments, the acylated or alkylated amino acid isa Dab, Orn, Lys, or homoLys residue and is located at position 40 of theC-terminally extended GIP agonist peptide or analog thereof.

In accordance with one embodiment, the GIP agonist peptide is modifiedto comprise an acyl group which is attached to the GIP agonist peptidevia an ester, thioester, or amide linkage for purposes of prolonginghalf-life in circulation and/or delaying the onset of and/or extendingthe duration of action and/or improving resistance to proteases such asDPP-IV.

In a specific aspect of the present disclosures, the GIP agonist peptideis modified to comprise an acyl group by direct acylation of an amine,hydroxyl, or thiol of a side chain of an amino acid of the GIP agonistpeptide. In some embodiments, the GIP agonist peptide is directlyacylated through the side chain amine, hydroxyl, or thiol of an aminoacid. In some embodiments, acylation is at position 10, 20, 24, 29, or40. In this regard, the acylated GIP agonist peptide can comprise theamino acid sequence of SEQ ID NO: 1, or a modified amino acid sequencethereof comprising one or more of the amino acid modifications describedherein, with at least one of the amino acids at positions 10, 20, 24,29, and 40 modified to any amino acid comprising a side chain amine,hydroxyl, or thiol. In some specific embodiments of the presentdisclosures, the direct acylation of the GIP agonist peptide occursthrough the side chain amine, hydroxyl, or thiol of the amino acid atposition 10 or 40.

In some embodiments, the amino acid comprising a side chain amine is anamino acid of Formula I:

In some exemplary embodiments, the amino acid of Formula I, is the aminoacid wherein n is 4 (Lys) or n is 3 (Orn).

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula II:

In some exemplary embodiments, the amino acid of Formula II is the aminoacid wherein n is 1 (Ser).

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula III:

In some exemplary embodiments, the amino acid of Formula II is the aminoacid wherein n is 1 (Cys).

In yet other embodiments, the amino acid comprising a side chain amine,hydroxyl, or thiol is a disubstituted amino acid comprising the samestructure of Formula I, Formula II, or Formula III, except that thehydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

In certain embodiments of the present disclosures, the acylated GIPagonist peptide comprises a spacer between the peptide and the acylgroup. In some embodiments, the GIP agonist peptide is covalently boundto the spacer, which is covalently bound to the acyl group.

The amino acid to which the spacer is attached can be any amino acid(e.g., a singly or doubly α-substituted amino acid) comprising a moietywhich permits linkage to the spacer. For example, an amino acidcomprising a side chain NH₂, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, orGlu) is suitable. In this respect, the acylated GIP agonist peptide cancomprise the amino acid sequence of SEQ ID NO: 1, or a modified aminoacid sequence thereof comprising one or more of the amino acidmodifications described herein, with at least one of the amino acids atpositions 10, 20, 24, 29, and 40 modified to any amino acid comprising aside chain amine, hydroxyl, or carboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol.

When acylation occurs through an amine group of a spacer the acylationcan occur through the alpha amine of the amino acid or a side chainamine. In the instance in which the alpha amine is acylated, the spaceramino acid can be any amino acid. For example, the spacer amino acid canbe a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, Ile, Trp, Met,Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoicacid, and 8-aminooctanoic acid. Alternatively, the spacer amino acid canbe an acidic residue, e.g., Asp and Glu.

In the instance in which the side chain amine of the spacer amino acidis acylated, the spacer amino acid is an amino acid comprising a sidechain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn). Inthis instance, it is possible for both the alpha amine and the sidechain amine of the spacer amino acid to be acylated, such that the GIPagonist peptide is diacylated. Embodiments of the present disclosuresinclude such diacylated molecules.

When acylation occurs through a hydroxyl group of a spacer, the aminoacid or one of the amino acids of the dipeptide or tripeptide can be anamino acid of Formula II. In a specific exemplary embodiment, the aminoacid is Ser.

When acylation occurs through a thiol group of a spacer, the amino acidor one of the amino acids of the dipeptide or tripeptide can be an aminoacid of Formula III. In a specific exemplary embodiment, the amino acidis Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. Incertain embodiments, the hydrophilic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophilic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophilic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophilic bifunctional spacer comprises a thiol group and acarboxylate. In specific embodiments, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments, the spacer is a hydrophobic bifunctional spacer.Hydrophobic bifunctional spacers are known in the art. See, e.g.,Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego,Calif., 1996), which is incorporated by reference in its entirety. Incertain embodiments, the hydrophobic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophobic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophobic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydropholic bifunctional spacer comprises a thiol group and acarboxylate. Suitable hydrophobic bifunctional spacers comprising acarboxylate, and a hydroxyl group or a thiol group are known in the artand include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid.

In some embodiments, the bifunctional spacer is not a dicarboxylic acidcomprising an unbranched, methylene of 1-7 carbon atoms between thecarboxylate groups. In some embodiments, the bifunctional spacer is adicarboxylic acid comprising an unbranched, methylene of 1-7 carbonatoms between the carboxylate groups.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional, or hydrophobic bifunctional spacer) in specificembodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or10 atoms) in length. In more specific embodiments, the spacer is about 3to 10 atoms (e.g., 6 to 10 atoms) in length and the acyl group is a C12to C18 fatty acyl group, e.g., C14 fatty acyl group, C16 fatty acylgroup, such that the total length of the spacer and acyl group is 14 to28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, or 28 atoms. In some embodiments, the length of the spacer andacyl group is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain foregoing embodiments, the bifunctionalspacer can be a synthetic or naturally occurring amino acid (including,but not limited to, any of those described herein) comprising an aminoacid backbone that is 3 to 10 atoms in length (e.g., 6-amino hexanoicacid, 5-aminovaleric acid, 7-aminoheptanoic acid, and 8-aminooctanoicacid). Alternatively, the spacer can be a dipeptide or tripeptide spacerhaving a peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) inlength. Each amino acid of the dipeptide or tripeptide spacer can be thesame as or different from the other amino acid(s) of the dipeptide ortripeptide and can be independently selected from the group consistingof: naturally-occurring and/or non-naturally occurring amino acids,including, for example, any of the D or L isomers of thenaturally-occurring amino acids (Ala, Cys, Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or any D or Lisomers of the non-naturally occurring amino acids selected from thegroup consisting of: β-alanine (β-Ala), N-α-methyl-alanine (Me-Ala),aminobutyric acid (Abu), δ-aminobutyric acid (γ-Abu), aminohexanoic acid(ε-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid,aminopiperidinecarboxylic acid, aminoserine (Ams),aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, β-aspartic acid (β-Asp), azetidine carboxylic acid,3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(O₂)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO₂)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), O-benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), and alkylated3-mercaptopropionic acid.

In some embodiments, the spacer comprises an overall negative charge,e.g., comprises one or two negatively charged amino acids, e.g., one ortwo acidic residues. In some embodiments, the dipeptide is not any ofthe dipeptides of general structure A-B, wherein A is selected from thegroup consisting of Gly, Gln, Ala, Arg, Asp, Asn, Ile, Leu, Val, Phe,and Pro, wherein B is selected from the group consisting of Lys, His,Trp.

In some exemplary embodiments, the GIP agonist peptide is modified tocomprise an acyl group by acylation of an amine, hydroxyl, or thiol of aspacer, which spacer is attached to a side chain of an amino acid atposition 10, 20, 24, 29, or 40, or at the C-terminal amino acid of theGIP agonist peptide.

In yet more specific embodiments, the acyl group is attached to theamino acid at position 10 or 40 of the GIP agonist peptide and,optionally, the length of the spacer and acyl group is 14 to 28 atoms.The amino acid at position 10 or 40, in some aspects, is an amino acidof Formula I, e.g., Lys, or a disubstituted amino acid related toFormula I. In more specific embodiments, the GIP agonist peptide lacksan intramolecular bridge, e.g., a covalent intramolecular bridge. TheGIP agonist peptide, for example, can be a peptide comprising one ormore alpha, alpha-disubstituted amino acids, e.g., AIB, for stabilizingthe alpha helix of the peptide. As shown herein, such peptidescomprising an acylated spacer covalently attached to the side chain ofthe amino acid at position 40 exhibit enhanced potency at the GIP,GLP-1, and glucagon receptors. Peptides comprising the same structureexcept further comprising an amino acid modification which selectivelyreduces activity at the glucagon receptor (e.g., substitution of Gln 3for Glu) are further contemplated herein.

Suitable methods of peptide acylation via amines, hydroxyls, and thiolsare known in the art. See, for example, Example 19 (for methods ofacylating through an amine), Miller, Biochem Biophys Res Commun 218:377-382 (1996); Shimohigashi and Stammer, Int J Pept Protein Res 19:54-62 (1982); and Previero et al., Biochim Biophys Acta 263: 7-13 (1972)(for methods of acylating through a hydroxyl); and San and Silvius, JPept Res 66: 169-180 (2005) (for methods of acylating through a thiol);Bioconjugate Chem. “Chemical Modifications of Proteins: History andApplications” pages 1, 2-12 (1990); Hashimoto et al., PharmacueticalRes. “Synthesis of Palmitoyl Derivatives of Insulin and their BiologicalActivity” Vol. 6, No: 2 pp. 171-176 (1989).

The acyl group of the acylated GIP agonist peptide can be of any size,e.g., any length carbon chain, and can be linear or branched. In somespecific embodiments of the present disclosures, the acyl group is a C4to C30 fatty acid. For example, the acyl group can be any of a C4 fattyacid, C6 fatty acid, C8 fatty acid, C10 fatty acid, C12 fatty acid, C14fatty acid, C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fattyacid, C24 fatty acid, C26 fatty acid, C28 fatty acid, or a C30 fattyacid. In some embodiments, the acyl group is a C8 to C20 fatty acid,e.g., a C14 fatty acid or a C16 fatty acid.

In an alternative embodiment, the acyl group is a bile acid. The bileacid can be any suitable bile acid, including, but not limited to,cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid,taurocholic acid, glycocholic acid, and cholesterol acid.

In some embodiments of the present disclosures, the GIP agonist peptideis modified to comprise an acyl group by acylation of a long chainalkane by the GIP agonist peptide. In specific aspects, the long chainalkane comprises an amine, hydroxyl, or thiol group (e.g.octadecylamine, tetradecanol, and hexadecanethiol) which reacts with acarboxyl group, or activated form thereof, of the GIP agonist peptide.The carboxyl group, or activated form thereof, of the GIP agonistpeptide can be part of a side chain of an amino acid (e.g., glutamicacid, aspartic acid) of the GIP agonist peptide or can be part of thepeptide backbone.

In certain embodiments, the GIP agonist peptide is modified to comprisean acyl group by acylation of the long chain alkane by a spacer which isattached to the GIP agonist peptide. In specific aspects, the long chainalkane comprises an amine, hydroxyl, or thiol group which reacts with acarboxyl group, or activated form thereof, of the spacer. Suitablespacers comprising a carboxyl group, or activated form thereof, aredescribed herein and include, for example, bifunctional spacers, e.g.,amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacersand hydrophobic bifunctional spacers.

As used herein, the term “activated form of a carboxyl group” refers toa carboxyl group with the general formula R(C═O)X, wherein X is aleaving group and R is the GIP agonist peptide or the spacer. Forexample, activated forms of a carboxyl groups may include, but are notlimited to, acyl chlorides, anhydrides, and esters. In some embodiments,the activated carboxyl group is an ester with a N-hydroxysuccinimideester (NHS) leaving group.

With regard to these aspects of the present disclosures, in which a longchain alkane is acylated by the GIP agonist peptide or the spacer, thelong chain alkane may be of any size and can comprise any length ofcarbon chain. The long chain alkane can be linear or branched. Incertain aspects, the long chain alkane is a C4 to C30 alkane. Forexample, the long chain alkane can be any of a C4 alkane, C6 alkane, C8alkane, C10 alkane, C12 alkane, C14 alkane, C16 alkane, C18 alkane, C20alkane, C22 alkane, C24 alkane, C26 alkane, C28 alkane, or a C30 alkane.In some embodiments, the long chain alkane comprises a C8 to C20 alkane,e.g., a C14 alkane, C16 alkane, or a C18 alkane.

Also, in some embodiments, an amine, hydroxyl, or thiol group of the GIPagonist peptide is acylated with a cholesterol acid. In specificembodiments, the GIP agonist peptide is linked to the cholesterol acidthrough a modified Cys spacer.

The acylated GIP agonist peptides described herein can be furthermodified to comprise a hydrophilic moiety. In some specific embodimentsthe hydrophilic moiety can comprise a polyethylene glycol (PEG) chain.The incorporation of a hydrophilic moiety can be accomplished throughany suitable means, such as any of the methods described herein. In thisregard, the acylated GIP agonist peptide can comprise SEQ ID NO: 1,including any of the modifications described herein, in which at leastone of the amino acids at position 10, 20, 24, 29, and 40 comprise anacyl group and at least one of the amino acids at position 16, 17, 21,24, 29, or 40, a position within a C-terminal extension, or theC-terminal amino acid are modified to a Cys, Lys, Orn, homo-Cys, orAc-Phe, and the side chain of the amino acid is covalently bonded to ahydrophilic moiety (e.g., PEG). In some embodiments, the acyl group isattached to position 10 or 40, optionally via a spacer comprising Cys,Lys, Orn, homo-Cys, or Ac-Phe, and the hydrophilic moiety isincorporated at a Cys residue at position 24.

Alternatively, the acylated GIP agonist peptide can comprise a spacer,wherein the spacer is both acylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Ac-Cys, Lys, Orn, homo-Cys, and Ac-Phe.

In a specific aspect of the present disclosures, the acylated GIPagonist peptide comprises the amino acid sequence of any of SEQ ID NOs:201-206, 213-215, 217-219, 223-225, 228-230, 232-234, 236-238, 241-245,248, 251, 252, 254, 256, 258, 260, 262, 263, 265, 266, 331, 334-339,357, and 358, and optionally, further comprises an amino acidmodification which selectively reduces activity at the glucagonreceptor, e.g., substitution of Gln 3 with Glu.

In accordance with some embodiments, the GIP agonist peptide is modifiedto comprise an alkyl group, e.g., an alkyl group which is notnaturally-occurring on an amino acid (e.g., an alkyl group which isnon-native to a naturally-occurring amino acid). Without being held toany particular theory, it is believed that alkylation of the GIP agonistpeptide of the present disclosures achieves similar, if not the same,effects as acylation of the GIP agonist peptides, e.g., a prolongedhalf-life in circulation, a delayed onset of action, an extendedduration of action, an improved resistance to proteases, such as DPP-IV,and increased potency at the GLP-1, GIP, and glucagon receptors.

Alkylation can be carried out at any positions within the GIP agonistpeptide, including any of positions 1-29, a position within a C-terminalextension, or the N- or C-terminal amino acid, provided that the GIPactivity (and optionally GLP-1 and/or glucagon activity) is retained, ifnot enhanced. Alkylation may occur, for example, at any amino acid whichis added to the amino acid sequence (SEQ ID NO: 1), e.g., at the N- orC-terminus. Nonlimiting examples include positions 1, 5, 10, 11, 12, 13,14, 16, 17, 18, 19, 20, 21, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The alkylgroup can be covalently linked directly to an amino acid of the GIPagonist peptide, or indirectly to an amino acid of the GIP agonistpeptide via a spacer, wherein the spacer is positioned between the aminoacid of the GIP agonist peptide and the alkyl group. GIP agonistpeptides may be alkylated at the same amino acid position where ahydrophilic moiety is linked, or at a different amino acid position.Nonlimiting examples include alkylation at position 10 or 40 andpegylation at one or more positions in the C-terminal portion of the GIPagonist peptide, e.g., position 24, 28 29, or 40, within a C-terminalextension, or at the C-terminus (e.g., through adding a C-terminal Cys).

In a specific aspect of the present disclosures, the GIP agonist peptideis modified to comprise an alkyl group by direct alkylation of an amine,hydroxyl, or thiol of a side chain of an amino acid of the GIP agonistpeptide. In some embodiments, the GIP agonist peptide is directlyalkylated through the side chain amine, hydroxyl, or thiol of an aminoacid. In some embodiments, alkylation is at position 10, 20, 24, 29, or40. In this regard, the alkylated GIP agonist peptide can comprise theamino acid sequence of SEQ ID NO: 1, or a modified amino acid sequencethereof comprising one or more of the amino acid modifications describedherein, with at least one of the amino acids at positions 10, 20, 24,29, and 40 modified to any amino acid comprising a side chain amine,hydroxyl, or thiol. In some specific embodiments of the presentdisclosures, the direct alkylation of the GIP agonist peptide occursthrough the side chain amine, hydroxyl, or thiol of the amino acid atposition 10.

In some embodiments, the amino acid comprising a side chain amine is anamino acid of Formula I. In some exemplary embodiments, the amino acidof Formula I, is the amino acid wherein n is 4 (Lys) or n is 3 (Orn).

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula II. In some exemplary embodiments, the aminoacid of Formula II is the amino acid wherein n is 1 (Ser).

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula III. In some exemplary embodiments, theamino acid of Formula III is the amino acid wherein n is 1 (Cys).

In yet other embodiments, the amino acid comprising a side chain amine,hydroxyl, or thiol is a disubstituted amino acid comprising the samestructure of Formula I, Formula II, or Formula III, except that thehydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

In one embodiment of the present disclosures, the alkylated GIP agonistpeptide comprises a spacer between the peptide and the alkyl group. Insome embodiments, the GIP agonist peptide is covalently bound to thespacer, which is covalently bound to the alkyl group. In some exemplaryembodiments, the GIP agonist peptide is modified to comprise an alkylgroup by alkylation of an amine, hydroxyl, or thiol of a spacer, whichspacer is attached to a side chain of an amino acid at position 10, 20,24, 29, or 40 of the GIP agonist peptide. The amino acid to which thespacer is attached can be any amino acid (e.g., a singly α-substitutedamino acid or an α,α-disubstituted amino acid) comprising a moiety whichpermits linkage to the spacer. For example, an amino acid comprising aside chain NH₂, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) issuitable. In this respect, the alkylated GIP agonist peptide cancomprise the amino acid sequence of SEQ ID NO: 1, or a modified aminoacid sequence thereof comprising one or more of the amino acidmodifications described herein, with at least one of the amino acids atpositions 10, 20, 24, 29, and 40 modified to any amino acid comprising aside chain amine, hydroxyl, or carboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol.

When alkylation occurs through an amine group of a spacer the alkylationcan occur through the alpha amine of the amino acid or a side chainamine. In the instance in which the alpha amine is alkylated, the spaceramino acid can be any amino acid. For example, the spacer amino acid canbe a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, Ile, Trp, Met,Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoicacid, and 8-aminooctanoic acid. Alternatively, the spacer amino acid canbe an acidic residue, e.g., Asp and Glu, provided that the alkylationoccurs on the alpha amine of the acidic residue. In the instance inwhich the side chain amine of the spacer amino acid is alkylated, thespacer amino acid is an amino acid comprising a side chain amine, e.g.,an amino acid of Formula I (e.g., Lys or Orn). In this instance, it ispossible for both the alpha amine and the side chain amine of the spaceramino acid to be alkylated, such that the GIP agonist peptide isdialkylated. Embodiments of the present disclosures include suchdialkylated molecules.

When alkylation occurs through a hydroxyl group of a spacer, the aminoacid or one of the amino acids of the dipeptide or tripeptide can be anamino acid of Formula II. In a specific exemplary embodiment, the aminoacid is Ser.

When acylation occurs through a thiol group of spacer, the amino acid orone of the amino acids of the dipeptide or tripeptide can be an aminoacid of Formula III. In a specific exemplary embodiment, the amino acidis Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. Incertain embodiments, the hydrophilic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophilic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophilic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophilic bifunctional spacer comprises a thiol group and acarboxylate. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments, the spacer is a hydrophobic bifunctional spacer. Incertain embodiments, the hydrophobic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophobic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydropholic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydropholic bifunctional spacer comprises a thiol group and acarboxylate. Suitable hydrophobic bifunctional spacers comprising acarboxylate, and a hydroxyl group or a thiol group are known in the artand include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional, or hydrophobic bifunctional spacer) in specificembodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or10 atoms)) in length. In more specific embodiments, the spacer is about3 to 10 atoms (e.g., 6 to 10 atoms) in length and the alkyl is a C12 toC18 alkyl group, e.g., C14 alkyl group, C16 alkyl group, such that thetotal length of the spacer and alkyl group is 14 to 28 atoms, e.g.,about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28atoms. In some embodiments, the length of the spacer and alkyl is 17 to28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain foregoing embodiments, the bifunctionalspacer can be a synthetic or non-naturally occurring amino acidcomprising an amino acid backbone that is 3 to 10 atoms in length (e.g.,6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and8-aminooctanoic acid). Alternatively, the spacer can be a dipeptide ortripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g.,6 to 10 atoms) in length. The dipeptide or tripeptide spacer can becomposed of naturally-occurring and/or non-naturally occurring aminoacids, including, for example, any of the amino acids taught herein. Insome embodiments, the spacer comprises an overall negative charge, e.g.,comprises one or two negatively charged amino acids, e.g., one or twoacidic residues. In some embodiments, the dipeptide spacer is selectedfrom the group consisting of: Ala-Ala, β-Ala-β-Ala, Leu-Leu, Pro-Pro,γ-aminobutyric acid-γ-aminobutyric acid, and γ-Glu-γ-Glu.

Suitable methods of peptide alkylation via amines, hydroxyls, and thiolsare known in the art. For example, a Williamson ether synthesis can beused to form an ether linkage between a hydroxyl group of the GIPagonist peptide and the alkyl group. Also, a nucleophilic substitutionreaction of the peptide with an alkyl halide can result in any of anether, thioether, or amino linkage.

The alkyl group of the alkylated GIP agonist peptide can be of any size,e.g., any length carbon chain, and can be linear or branched. In someembodiments of the present disclosures, the alkyl group is a C4 to C30alkyl. For example, the alkyl group can be any of a C4 alkyl, C6 alkyl,C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28 alkyl, or a C30 alkyl. Insome embodiments, the alkyl group is a C8 to C20 alkyl, e.g., a C14alkyl or a C16 alkyl.

In some specific embodiments, the alkyl group comprises a steroid moietyof a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholicacid, lithocholic acid, taurocholic acid, glycocholic acid, andcholesterol acid.

In some embodiments of the present disclosures, the GIP agonist peptideis modified to comprise an alkyl group by reacting a nucleophilic, longchain alkane with the GIP agonist peptide, wherein the GIP agonistpeptide comprises a leaving group suitable for nucleophilicsubstitution. In specific aspects, the nucleophilic group of the longchain alkane comprises an amine, hydroxyl, or thiol group (e.g.octadecylamine, tetradecanol, and hexadecanethiol). The leaving group ofthe GIP agonist peptide can be part of a side chain of an amino acid orcan be part of the peptide backbone. Suitable leaving groups include,for example, N-hydroxysuccinimide, halogens, and sulfonate esters.

In certain embodiments, the GIP agonist peptide is modified to comprisean alkyl group by reacting the nucleophilic, long chain alkane with aspacer which is attached to the GIP agonist peptide, wherein the spacercomprises the leaving group. In specific aspects, the long chain alkanecomprises an amine, hydroxyl, or thiol group. In certain embodiments,the spacer comprising the leaving group can be any spacer discussedherein, e.g., amino acids, dipeptides, tripeptides, hydrophilicbifunctional spacers and hydrophobic bifunctional spacers furthercomprising a suitable leaving group.

With regard to these aspects of the present disclosures, in which a longchain alkane is alkylated by the GIP agonist peptide or the spacer, thelong chain alkane may be of any size and can comprise any length ofcarbon chain. The long chain alkane can be linear or branched. Incertain aspects, the long chain alkane is a C4 to C30 alkane. Forexample, the long chain alkane can be any of a C4 alkane, C6 alkane, C8alkane, C10 alkane, C12 alkane, C14 alkane, C16 alkane, C18 alkane, C20alkane, C22 alkane, C24 alkane, C26 alkane, C28 alkane, or a C30 alkane.In some embodiments, the long chain alkane comprises a C8 to C20 alkane,e.g., a C14 alkane, C16 alkane, or a C18 alkane.

Also, in some embodiments, alkylation can occur between the GIP agonistpeptide and a cholesterol moiety. For example, the hydroxyl group ofcholesterol can displace a leaving group on the long chain alkane toform a cholesterol-GIP agonist peptide product.

The alkylated GIP agonist peptides described herein can be furthermodified to comprise a hydrophilic moiety. In some specific embodimentsthe hydrophilic moiety can comprise a polyethylene glycol (PEG) chain.The incorporation of a hydrophilic moiety can be accomplished throughany suitable means, such as any of the methods described herein. In thisregard, the alkylated GIP agonist peptide can comprise SEQ ID NO: 1, ora modified amino acid sequence thereof comprising one or more of theamino acid modifications described herein, in which at least one of theamino acids at position 10, 20, 24, 29, and 40 comprise an alkyl groupand at least one of the amino acids at position 16, 17, 21, 24, 29, and40, a position within a C-terminal extension or the C-terminal aminoacid are modified to a Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the sidechain of the amino acid is covalently bonded to a hydrophilic moiety(e.g., PEG). In some embodiments, the alkyl group is attached toposition 10 or 40, optionally via a spacer comprising Cys, Lys, Orn,homo-Cys, or Ac-Phe, and the hydrophilic moiety is incorporated at a Cysresidue at position 24.

Alternatively, the alkylated GIP agonist peptide can comprise a spacer,wherein the spacer is both alkylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

Modifications that Improve DPP-IV Resistance

In some aspects of the present disclosures, the GIP agonist peptidecomprises one or two modifications at position 1 and/or 2 which increasethe peptide's resistance to dipeptidyl peptidase IV (DPP IV) cleavage.In exemplary embodiments, the amino acid at position 2 of the GIPagonist peptide is substituted with one of: D-serine, D-alanine, valine,glycine, N-methyl serine, N-methyl alanine, or amino isobutyric acid(AIB). In exemplary embodiments, the amino acid at position 1 of the GIPagonist peptide is substituted with one of: D-histidine,desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine,N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, oralpha, alpha-dimethyl imidiazole acetic acid (DMIA). In some aspects,the GIP agonist peptide comprising an amino acid modification whichincreases resistance to DPP IV further comprises an intramolecularbridge or alpha, alpha di-substituted amino acid, and optionally anamino acid modification which selectively reduces the activity at theglucagon receptor, such as, for example, a substitution of Gln3 withGlu.

Modifications that Reduce Degradation

In exemplary embodiments, any of the GIP agonist peptides of the presentdisclosures can be further modified to improve stability of the peptideby modifying the amino acid at position 15 and/or 16 of SEQ ID NO: 1 toreduce degradation of the peptide over time, especially in acidic oralkaline buffers. Such modifications reduce cleavage of the Asp15-Ser16peptide bond. In exemplary embodiments, the amino acid modification atposition 15 is a deletion or substitution of Asp with glutamic acid,homoglutamic acid, cysteic acid or homocysteic acid. In other exemplaryembodiments, the amino acid modification at position 16 is a deletion orsubstitution of Ser with Thr or AIB. In alternative or additionalembodiments, Ser at position 16 is substituted with glutamic acid orwith another negatively charged amino acid having a side chain with alength of 4 atoms, or alternatively with any one of glutamine,homoglutamic acid, or homocysteic acid. Such modifications can reducedegradation or cleavage at a pH within the range of 5.5 to 8, forexample, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, upto 100% of the original peptide after 24 hours at 25° C. Suchmodifications reduce cleavage of the peptide bond between Asp15-Ser16.

In some embodiments, the GIP agonist peptide comprises a modificationwhich prevents oxidative degradation of the peptide. In some aspects,the methionine residue at position 27 of the native glucagon peptide ismodified, e.g. by deletion or substitution. In some embodiments, the Metat position 27 is substituted with leucine, isoleucine or norleucine. Insome specific embodiments, Met at position 27 is substituted withleucine or norleucine.

In some embodiments, the GIP agonist peptide comprises one or moremodifications that reduce degradation through deamidation of Gln. Insome aspects, the Gln at position 20 and/or 24 of the GIP agonistpeptide is modified, e.g. by deletion or substitution. In someembodiments, the Gln at position 20 and/or 24 of the GIP agonist peptideis substituted with Ser, Thr, Ala or AIB. In some embodiments the Gln atposition 20 and/or 24 of the GIP agonist peptide is substituted withLys, Arg, Orn, or Citrulline.

In some embodiments, the GIP agonist peptide comprises an amino acidmodification which reduces degradation through dehydration of Asp toform a cyclic succinimide intermediate followed by isomerization toiso-aspartate. Accordingly, in some aspects, the Asp at position 21 ofthe GIP agonist peptide is modified, e.g. by deletion or substitution.In some embodiments, position 21 of the GIP agonist peptide issubstituted with Glu, homoglutamic acid or homocysteic acid. In somespecific embodiments, position 21 is substituted with Glu.

Modifications that Enhance Solubility

In another embodiment, the solubility of any of the GIP agonist peptidesis improved by one or more amino acid substitutions and/or additionsthat introduce a charged amino acid into the C-terminal portion of thepeptide, preferably at a position C-terminal to position 27 of SEQ IDNO: 1. Optionally, one, two or three charged amino acids are introducedwithin the C-terminal portion, preferably C-terminal to position 27. Insome embodiments of the present disclosures, the native amino acid(s) atpositions 28 and/or 29 are substituted with one or two charged aminoacids, and/or in further embodiments one to three charged amino acidsare also added to the C-terminus of the GIP agonist peptide. Inexemplary embodiments, one, two or all of the charged amino acids arenegative-charged or acidic amino acids. In some embodiments, thenegative-charged or acidic amino acids are aspartic acid or glutamicacid. In other embodiments, one, two, three or all of the charged aminoacids are positively charged. Such modifications increase solubility,e.g. provide at least 2-fold, 5-fold, 10-fold, 15-fold, 25-fold, 30-foldor greater solubility relative to native glucagon at a given pH betweenabout 5.5 and 8, e.g., pH 7, when measured after 24 hours at 25° C.

The addition of a hydrophilic moiety also can enhance solubility of theGIP agonist peptide. Hydrophilic moieties and conjugation thereof topeptides is further described herein. See, “Conjugates.” In exemplaryembodiments, the GIP agonist peptide is conjugated to a hydrophilicmoiety, e.g., polyethylene glycol, at position 16, 17, 20, 21, 24 or 29of the GIP agonist peptide, within a C-terminal extension, and/or at theC-terminal amino acid of the peptide. Such modifications also enhancethe duration of action or half-life of the peptide in circulation.

Other Modifications

Additional modifications, e.g. conservative substitutions, may be madeto the GIP agonist peptide that still allow it to retain GIP activity(and optionally GLP-1 activity and/or glucagon activity). Exemplarymodifications include but are not limited to the following:

Non-conservative or conservative substitutions, additions or deletionsthat do not substantially affect activity, for example, conservativesubstitutions at one or more of positions 2, 5, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29; substitution of one ormore of these positions with Ala; deletion of amino acids at one or moreof positions 27, 28 or 29; or deletion of amino acid 29 optionallycombined with a C-terminal amide or ester in place of the C-terminalcarboxylic acid group; substitution of Lys at position 12 with Arg;substitution of Tyr at position 10 with Val or Phe;

Preservation of activity after pegylation is provided by the addition ofGPSSGAPPPS (SEQ ID NO: 3) to the C-terminus.

In some embodiments, position 18 is substituted with an amino acidselected from the group consisting of Ala, Ser, or Thr. In someembodiments the amino acid at position 20 is substituted with Ser, Thr,Lys, Arg, Orn, Citrulline or AIB. In some embodiments, position 21 issubstituted with Glu, homoglutamic acid or homocysteic acid. In someembodiments, the GIP agonist peptide comprises 1 to 10 amino acidmodifications selected from positions 16, 17, 18, 20, 21, 23, 24, 27, 28and 29. In exemplary embodiments, the modifications are one or moreamino acid substitutions selected from the group consisting of Gln17,Ala18, Glu21, Ile23, Ala24, Val27 and Gly29. In some embodiments, 1 to 5amino acids selected from positions 17-26 differ from the parentpeptide. In other embodiments, 1 to 5 amino acids selected frompositions 17-24 differ from the parent peptide. In yet otherembodiments, the modifications are Gln17, Ala18, Glu21, Ile23 and Ala24.

In some embodiments, one or more amino acids is added to the carboxyterminus of the GIP agonist peptide. The amino acid is typicallyselected from one of the 20 common amino acids, and in some embodimentsthe amino acid has an amide group in place of the carboxylic acid of thenative amino acid. In exemplary embodiments the added amino acid isselected from the group consisting of glutamic acid and aspartic acidand glycine.

Other modifications that do not destroy activity include W10 or R20.

In some embodiments, the GIP agonist peptides disclosed herein aremodified by truncation of the C-terminus by one or two amino acidresidues yet retain similar activity and potency at the glucagon, GLP-1and/or GIP receptors. In this regard, the amino acid at position 29and/or 28 can be deleted.

Activity of the Glucagon Antagonist Peptide

Glucagon Receptor Antagonism

In some embodiments of the present disclosures, the glucagon antagonistpeptide exhibits at least or about 60% inhibition of the maximumresponse of native glucagon at the glucagon receptor. In exemplaryembodiments, the glucagon antagonist peptide exhibits at least or about65%, at least or about 70%, at least or about 75%, at least or about80%, at least or about 85%, at least or about 90%, at least or about95%, or at least or about 100% inhibition of the maximum response ofnative glucagon at the glucagon receptor. Accordingly, the glucagonantagonist peptide binds to the glucagon receptor and counteractsglucagon activity or prevents glucagon function.

In some aspects of the present disclosures, the glucagon antagonistpeptide has an IC50 at the glucagon receptor which is in the micromolarrange. In exemplary embodiments, the IC50 of the glucagon antagonistpeptide at the glucagon receptor is less than 1000 μM, less than 900 μM,less than 800 μM, less than 700 μM, less than 600 μM, less than 500 μM,less than 400 μM, less than 300 μM, less than 200 μM. In someembodiments, the IC50 of the glucagon antagonist peptide at the glucagonreceptor is about 100 μM or less, e.g., about 75 μM or less, about 50 μMor less, about 25 μM or less, about 10 μM or less, about 8 μM or less,about 6 μM or less, about 5 μM or less, about 4 μM or less, about 3 μMor less, about 2 μM or less, or about 1 μM or less.

In some aspects of the present disclosures, the glucagon antagonistpeptide has an IC50 at the glucagon receptor which is in the nanomolarrange. In exemplary embodiments, the IC50 of the glucagon antagonistpeptide at the glucagon receptor is less than 1000 nM, less than 900 nM,less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM,less than 400 nM, less than 300 nM, less than 200 nM. In someembodiments, the IC50 of the glucagon antagonist peptide at the glucagonreceptor is about 100 nM or less, e.g., about 75 nM or less, about 50 nMor less, about 25 nM or less, about 10 nM or less, about 8 nM or less,about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nMor less, about 2 nM or less, or about 1 nM or less. In some aspects, theIC50 of the glucagon antagonist at the glucagon receptor is between 0.1nM and 500 nM. In some aspects, the IC50 is about 0.1 nM or about 500nM. In some embodiments, the glucagon antagonist peptide exhibits anIC50 for glucagon receptor activation which is in the picomolar range.In exemplary embodiments, the IC50 of the glucagon antagonist peptide atthe glucagon receptor is less than 1000 pM, less than 900 pM, less than800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than400 pM, less than 300 pM, less than 200 pM. In some embodiments, theIC50 of the glucagon antagonist peptide at the glucagon receptor isabout 100 μM or less, e.g., about 75 pM or less, about 50 pM or less,about 25 pM or less, about 10 pM or less, about 8 pM or less, about 6 pMor less, about 5 pM or less, about 4 pM or less, about 3 pM or less,about 2 pM or less, or about 1 pM or less.

In some embodiments, the glucagon antagonist peptide is a glucagonantagonist which, at a concentration of about 1 μM, exhibits less thanor about 20% of the maximum agonist activity achieved by glucagon at theglucagon receptor. In some embodiments, the glucagon antagonist peptideis a glucagon antagonist which exhibits less than or about 15%, lessthan or about 10%, less than or about 5%, less than or about 1%, orabout 0% of the maximum agonist activity achieved by glucagon at theglucagon receptor, when the peptide present at a concentration of about1 μM.

In some aspects, the glucagon antagonist peptide is a “full antagonist”at the glucagon receptor and in other aspects, the glucagon antagonistpeptide is a “partial antagonist” at the glucagon receptor By “fullantagonist” as used herein is meant an antagonist that binds to thereceptor and does not exhibit any agonist activity at the receptor itantagonizes. The term “partial antagonist” as used herein is synonymouswith “partial agonist” which is a compound that exhibits a lower amountof agonist activity at a receptor as compared to a full agonist, but thepartial agonist serves as an antagonist since its occupation of areceptor prevents the full agonist from binding, thereby producing a netdecrease in the receptor activation as compared to the level of receptoractivation if all receptors were bound by full agonists.

In some aspects of the present disclosures, the glucagon antagonistpeptide exhibits activity (agonist or antagonist) at only one receptor.Accordingly, the glucagon antagonist peptide is some aspects is a “pureglucagon antagonist” and does not produce any detected stimulation ofthe glucagon receptor or any other receptor, including, e.g., the GLP-1receptor, the GIP receptor, as measured by cAMP production using avalidated in vitro model assay, such as that described in Example 2. Forexample, a pure glucagon antagonist exhibits less than about 5% (e.g.,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%, about 0%) of the maximum agonist activity achieved by glucagonat the glucagon receptor and exhibits less than about 5% (e.g., lessthan about 4%, less than about 3%, less than about 2%, less than about1%, and about 0%) of the maximum agonist activity achieved by GLP-1 atthe GLP-1 receptor and/or exhibits less than about 5% (e.g., less thanabout 4%, less than about 3%, less than about 2%, less than about 1%,and about 0%) of the maximum agonist activity achieved by GIP at the GIPreceptor.

In other embodiments of the present disclosures, the glucagon antagonistpeptide exhibits activity (agonist or antagonist) at more than onereceptor. In such embodiments, the glucagon antagonist peptide has lostselectivity for one receptor over another. For example, the glucagonantagonist peptide in some embodiments is a glucagon receptor antagonistand an antagonist or agonist at another receptor, e.g., GLP-1 receptorand/or GIP receptor. The glucagon antagonist peptide in some embodimentsexhibits mixed properties insofar as it exhibits antagonist activity atthe glucagon receptor and agonist activity at another receptor, e.g.,the GLP-1 receptor, the GIP receptor. By way of example, the glucagonantagonist peptide in some aspects exhibits both antagonist activity atthe glucagon receptor and agonist activity at the GLP-1 receptor(“Glucagon receptor antagonist/GLP-1 receptor agonists”). In someaspects, the glucagon antagonist peptide has any of the IC50s at theglucagon receptor described herein and has any of the EC50s at the GLP-1receptor described herein. In some embodiments, the IC50 of the glucagonantagonist peptide at the glucagon receptor is less than or about50-fold, less than or about 40-fold, less than or about 30-fold, or lessthan or about 20-fold different (higher or lower) from its EC50 at theGLP-1 receptor. In some embodiments, the ratio of the IC50 of theglucagon antagonist peptide at the glucagon receptor divided by the EC50of the glucagon antagonist peptide at the GLP-1 receptor is less thanabout 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. Insome embodiments, the ratio of the EC50 of the glucagon antagonistpeptide at the GLP-1 receptor divided by the IC50 of the glucagonantagonist peptide at the glucagon receptor is less than about 100, 75,60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1.

Activity of Conjugates

In some embodiments, the glucagon antagonist peptides described hereinexhibit inhibitory activity at the glucagon receptor and/or agonistactivity at the GLP-1 receptor as described above and, when the glucagonantagonist peptide is part of a conjugate (e.g., is conjugated to aheterologous moiety, e.g., a hydrophilic moiety, e.g., a polyethyleneglycol), the glucagon antagonist peptide exhibits an activity that islower (i.e. lower inhibitory potency or higher IC50) than when theglucagon antagonist peptide is not part of the conjugate. In someaspects, the glucagon antagonist peptide when not part of conjugateexhibits an inhibitory potency at the glucagon receptor that is about10-fold or greater than the potency of the glucagon antagonist peptidewhen part of a conjugate. In some aspects, the glucagon antagonistpeptide when unconjugated exhibits an inhibitory potency at the glucagonreceptor that is about 10-fold, about 15-fold, about 20-fold, about25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold,about 50-fold, about 100-fold, or even greater-fold the potency of theglucagon antagonist peptide when conjugated.

Structure of the Glucagon Antagonist Peptide

In some embodiments of the present disclosures, the glucagon antagonistpeptide is a glucagon antagonist, which exhibits any of the activities(potency or EC50) at the indicated receptor as described above, and isstructurally similar to native human glucagon (SEQ ID NO: 1), e.g., isan analog of native human glucagon (or a glucagon analog). Such analogsof glucagon exhibiting glucagon receptor antagonist activity are knownin the art. For example, glucagon antagonists in which one or more aminoacids of the native human glucagon amino acid sequence were deleted orsubstituted include: [des His¹] [Glu⁹]-glucagon amide (Unson et al.,(1989) Peptides 10, 1171; Post et al., (1993) Proc. Natl. Acad. Sci. USA90, 1662), des His¹, Phe⁶ [Glu⁹]-glucagon amide (Azizh et al., (1995)Bioorg. & Med. Chem. Lett. 16, 1849) and Nle⁹, Ala^(11,16)-glucagonamide (Unson et al. (1994) J. Biol. Chem. 269(17), 12548). Otheranalogues include substitutions at positions 4 (Ahn J M et al. (2001) J.Pept. Res. 58(2):151-8), 1 (Dharanipragada, R. et al. (1993) Int. J.Pept. Res. 42(1): 68-77) and at position 4, 5, 12, 17 and 18 of theglucagon sequence (Gysin B et al. 1986. Biochemistry. 25(25):8278-84).Furthermore, glucagon antagonists which are structurally similar tonative human glucagon are also described in International PatentApplication Publication Nos. WO 2009/058662 and WO 2009/058734 and U.S.Application Nos. 60/983,783; 60/983,766; and 61/090,441; the contents ofwhich are incorporated by reference in their entirety.

Accordingly, in some embodiments, the glucagon antagonist peptide is ananalog of native human glucagon (SEQ ID NO: 1) which comprises an aminoacid sequence based on SEQ ID NO: 1 but is modified with 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in some instances, 16 or more(e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.) amino acidmodifications. In some embodiments, the glucagon antagonist peptidecomprises a total of 1, up to 2, up to 3, up to 4, up to 5, up to 6, upto 7, up to 8, up to 9, or up to 10 amino acid modifications relative tothe native human glucagon sequence (SEQ ID NO: 1). In some embodiments,the modifications are any of those described herein, e.g., truncation atthe N-terminus, formation into depsipeptide, substitution at position 9,acylation, alkylation, pegylation, truncation at C-terminus,substitution of the amino acid at one or more of positions 1, 2, 3, 7,10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29.

In some embodiments, the glucagon antagonist peptide of the presentdisclosures comprises an amino acid sequence which has at least 25%sequence identity to the amino acid sequence of native human glucagon(SEQ ID NO: 1). In some embodiments, the glucagon antagonist peptidecomprises an amino acid sequence which is at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90% or has greater than 90% sequence identity to SEQ ID NO: 1. Insome embodiments, the amino acid sequence of the glucagon antagonistpeptide which has the above-referenced % sequence identity is thefull-length amino acid sequence of the glucagon antagonist peptide. Insome embodiments, the amino acid sequence of the glucagon antagonistpeptide which has the above-referenced % sequence identity is only aportion of the amino acid sequence of the glucagon antagonist peptide.In some embodiments, the glucagon antagonist peptide comprises an aminoacid sequence which has about A % or greater sequence identity to areference amino acid sequence of at least 5 contiguous amino acids(e.g., at least 6, at least 7, at least 8, at least 9, at least 10 aminoacids) of SEQ ID NO: 1, wherein the reference amino acid sequence beginswith the amino acid at position C of SEQ ID NO: 1 and ends with theamino acid at position D of SEQ ID NO: 1, wherein A is 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,99; C is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28or 29. Any and all possible combinations of the foregoing parameters areenvisioned, including but not limited to, e.g., wherein A is 90% and Cand D are 1 and 27, or 6 and 27, or 8 and 27, or 10 and 27, or 12 and27, or 16 and 27.

The GIP agonist peptides which are analogs of native human glucagon (SEQID NO: 1) described herein may comprise a peptide backbone of any numberof amino acids, i.e., can be of any peptide length. In some embodiments,the GIP agonist peptides described herein are the same length as SEQ IDNO: 1, i.e., are 29 amino acids in length. In some embodiments, the GIPagonist peptide is longer than 29 amino acids in length, e.g., the GIPagonist peptide comprises a C-terminal extension of 1-21 amino acids, asfurther described herein. Accordingly, the GIP agonist peptide in someembodiments, is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 amino acids in length. In someembodiments, the GIP agonist peptide is longer than 29 amino acids inlength (e.g., greater than 50 amino acids, (e.g., at least or about 60,at least or about 70, at least or about 80, at least or about 90, atleast or about 100, at least or about 150, at least or about 200, atleast or about 250, at least or about 300, at least or about 350, atleast or about 400, at least or about 450, at least or about 500 aminoacids in length) due to fusion with another peptide. In otherembodiments, the GIP agonist peptide is less than 29 amino acids inlength, e.g., 28, 27, 26, 25, 24, 23, amino acids.

In accordance with the foregoing, in some aspects, the glucagonantagonist peptide of the present disclosures is an analog of nativehuman glucagon (SEQ ID NO: 1) comprising SEQ ID NO: 1 modified with oneor more amino acid modifications which reduce or destroy glucagonactivity, which increase or enhance GLP-1 activity or GIP activity,enhance stability, e.g., by reducing degradation of the peptide (e.g.,by improving resistance to DPP-IV proteases), enhance solubility,increase half-life, delay the onset of action, extend the duration ofaction at the GIP, glucagon, or GLP-1 receptor, or a combination of anyof the foregoing. Such amino acid modifications, in addition to othermodifications, are further described herein.

Exemplary Embodiments of the Glucagon Antagonist Peptide

Under normal circumstances, native human glucagon activates the glucagonreceptor in the human body. Described herein are modifications of thenative human glucagon amino acid sequence (SEQ ID NO: 1) which alterthis hormone, such that is antagonizes (e.g., binds to but does notactivate downstream signaling through) the glucagon receptor.

In some embodiments of the present disclosures, the glucagon antagonistpeptide comprises an amino acid sequence based on the sequence of nativehuman glucagon (SEQ ID NO: 1) but is modified by the deletion of thefirst two to five amino acid residues from the N-terminus andsubstitution of the aspartic acid residue at position nine of the nativeprotein (SEQ ID NO: 1) with a glutamic acid, homoglutamic acid,β-homoglutamic acid, a sulfonic acid derivative of cysteine, or analkylcarboxylate derivative of cysteine having the structure of:

wherein X₅ is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl.

In specific aspects, the glucagon antagonist peptide exhibiting glucagonantagonist activity and comprising the deletion of two to five aminoacid residues from the N-terminus and substitution of the Asp atposition 9 of the native glucagon, is further modified by up to threeamino acid modifications. For example, the glucagon antagonist peptidein some aspects comprise one, two, or three conservative amino acidmodifications. Alternatively or additionally, the glucagon antagonistpeptide in some aspects comprises one or more amino acid modificationsselected from the group consisting of:

-   -   A. substitution of one or two amino acids at positions 10, 20,        and 24, (according to the amino acid numbering of SEQ ID NO: 1),        or the N- or C-terminal amino acid of the glucagon antagonist        with an amino acid covalently attached to an acyl group or alkyl        group via an ester, ether, thioether, amide, or alkyl amine        linkage;    -   B. substitution of one or two amino acids at positions 16, 17,        20, 21, and 24 (according to the amino acid numbering of SEQ ID        NO: 1), or the N- or C-terminal amino acid of the glucagon        antagonist with an amino acid selected from the group consisting        of: Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine        (Ac-Phe), wherein the amino acid of the group is covalently        bonded to a hydrophilic moiety;    -   C. addition of an amino acid covalently bonded to a hydrophilic        moiety to the N- or C-terminus of the glucagon antagonist;    -   D. substitution of Asp at position 15 (according to the        numbering of SEQ ID NO: 1) with cysteic acid, glutamic acid,        homoglutamic acid, and homocysteic acid;    -   E. substitution of Ser at position 16 (according to the        numbering of SEQ ID NO: 1) with cysteic acid, glutamic acid,        homoglutamic acid, and homocysteic acid;    -   F. substitution with AIB at one or more of positions 16, 20, 21,        and 24 according to the amino acid numbering of SEQ ID NO: 1;    -   G. deletion of the amino acid at position 29 or the amino acids        at positions 28 and 29, according to the numbering of SEQ ID NO:        1;    -   H. substitution of each or both of the Asn at position 28 and        the Thr at position 29 (according to the amino acid numbering of        SEQ ID NO: 1) with charged amino acids; and/or addition of one        to two charged amino acids at the C-terminus of SEQ ID NO: 1;    -   I. substitution of the Met at position 27 (according to the        numbering of SEQ ID NO: 1) with Leu or norleucine;    -   J. addition of a peptide having the amino acid sequence of any        of SEQ ID NOs: 1119-1121 and 1153 to the C-terminus of SEQ ID        NO: 1; wherein Thr at position 29 (according to the numbering of        SEQ ID NO: 1) is Thr or Gly; and    -   K. replacement of the C-terminal carboxylate with an amide or        ester.

In specific aspects of the present disclosures, the glucagon antagonistpeptide comprises an amino acid modification of A, B, or C, as describedabove, or a combination thereof. In yet other specific embodiments, theglucagon antagonist peptide further comprises an amino acid modificationof any of D to K as described above, or a combination thereof, inaddition to the amino acid modification(s) of A, B, and/or C.

In some embodiments, the glucagon antagonist peptide comprises the aminoacid sequence of native human glucagon in which the first 5 amino acidshave been removed from the N-terminus, and the remaining N-terminalalpha amino group has been replaced with a hydroxyl group. TheN-terminal residue of these embodiments is phenyl lactic acid (PLA).

In certain aspects of the present disclosures in which the first 5 aminoacids have been removed from the N-terminus, and the remainingN-terminal amino group has been replaced with a hydroxyl group, theamino acid at position 9 (according to the numbering of SEQ ID NO: 1) ismodified by substituting the aspartic acid residue at position four(position 9 of the native glucagon) with an amino acid of the generalstructure:

wherein X₆ is C₁-C₃ alkyl, C₂-C₃ alkenyl or C₂-C₃ alkynyl. In someembodiments, X is C₁-C₃ alkyl, and in other embodiments, X is C₂ alkyl.In some embodiments, the glucagon antagonist peptide comprises SEQ IDNO: 1 in which the first 5 amino acids have been deleted from theN-terminus, and the aspartic acid residue at position four (position 9of the native glucagon) has been substituted with cysteic acid orhomocysteic acid. However, substitution at position 9 (according to thenumbering of SEQ ID NO: 1) is considered optional in embodiments inwhich PLA is the N-terminal residue, since the modification at position9 is not required for antagonist activity at the glucagon receptor.

In certain aspects of the present disclosures, the glucagon antagonistpeptide comprises SEQ ID NO: 1 in which the first five amino acids ofthe N-terminus has been deleted and the 6^(th) residue of SEQ ID NO: 1(which is the 1^(st) amino acid of the glucagon antagonist peptide) isPLA or other phenylalanine analog, including 3, 4-2F-phenylalanine (3,4-2F-Phe), 2-naphthyalanine (2-Nal), N-acyl-phenylalanine (Ac-Phe),alpha-methylhydrocinnamic acid (MCA) and benzylmalonic acid (BMA), forexample. However, as shown in WO 2009/058662, substitution with PLA atposition 6 (according to the numbering of SEQ ID NO: 1) provides a morepotent glucagon antagonist.

In certain aspects of the present disclosures, the glucagon antagonistpeptide comprises the general structure of A-B-C, wherein A is selectedfrom the group consisting of:

-   -   (i) phenyl lactic acid (PLA);    -   (ii) an oxy derivative of PLA;    -   (iii) a peptide of 2 to 6 amino acids in which two consecutive        amino acids of the peptide are linked via an ester or ether        bond;

B represents amino acids i to 26 of SEQ ID NO: 1, wherein i is 3, 4, 5,6, or 7, optionally comprising one or more amino acid modificationsselected from the group consisting of:

-   -   (iv) Asp at position 9 (according to the amino acid numbering of        SEQ ID NO: 1) is substituted with a Glu, a sulfonic acid        derivative of Cys, homoglutamic acid, β-homoglutamic acid, or an        alkylcarboxylate derivative of cysteine having the structure of:

wherein X₅ is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl.

-   -   (v) substitution of one or two amino acids at positions 10, 20,        and 24, (according to the amino acid numbering of SEQ ID NO: 1)        with an amino acid covalently attached to an acyl or alkyl group        via an ester, ether, thioether, amide, or alkyl amine linkage;    -   (vi) substitution of one or two amino acids at positions 16, 17,        20, 21, and 24 (according to the amino acid numbering of SEQ ID        NO: 1) with an amino acid selected from the group consisting of:        Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine        (Ac-Phe), wherein the amino acid of the group is covalently        attached to a hydrophilic moiety;    -   (vii) Asp at position 15 (according to the numbering of SEQ ID        NO: 1) is substituted with cysteic acid, glutamic acid,        homoglutamic acid, and homocysteic acid;    -   (viii) Ser at position 16 (according to the numbering of SEQ ID        NO: 1) is substituted with cysteic acid, glutamic acid,        homoglutamic acid, and homocysteic acid;    -   (ix) substitution with AIB at one or more of positions 16, 20,        21, and 24 according to the amino acid numbering of SEQ ID NO:        1;

and C is selected from the group consisting of:

-   -   (x) X;    -   (xi) X-Y;    -   (xii) X-Y-Z; and    -   (xiii) X-Y-Z-R10,        wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid;        Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl        phenylalanine (Ac-Phe), or a charged amino acid; wherein R10 is        selected from a group consisting of SEQ ID NOs: 1119-1121 and        1153; and    -   (xiv) any of (x) to (xiii) in which the C-terminal carboxylate        is replaced with an amide.

In specific aspects, the glucagon antagonist peptide comprises thegeneral structure A-B-C as described herein and exhibits agonistactivity at the GLP-1 receptor. Accordingly, in some aspects, theglucagon antagonist peptide comprises (1) a stabilized alpha helixthrough means described herein (e.g., through an intramolecular bridge,or incorporation of one or more alpha, alpha-di-substituted amino acids,or an acidic amino acid at position 16 (according to the numbering ofSEQ ID NO:1), or a combination thereof; (2) a C-terminal amide or esterin place of a C-terminal carboxylate, and (3) a general structure ofA-B-C,

wherein A is selected from the group consisting of

-   -   (i) PLA;    -   (ii) an oxy derivative of PLA; and    -   (iii) a peptide of 2 to 6 amino acids in which two consecutive        amino acids of the peptide are linked via an ester or ether        bond;

wherein B represents amino acids p to 26 of SEQ ID NO: 1, wherein p is3, 4, 5, 6, or 7, optionally comprising one or more amino acidmodifications selected from the group consisting of:

-   -   (iv) Asp at position 9 (according to the amino acid numbering of        SEQ ID NO: 1) is substituted with a Glu, a sulfonic acid        derivative of Cys, homoglutamic acid, β-homoglutamic acid, or an        alkylcarboxylate derivative of cysteine having the structure of:

wherein X₅ is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl;

-   -   (v) substitution of one or two amino acids at positions 10, 20,        and 24, (according to the amino acid numbering of SEQ ID NO: 1)        with an amino acid covalently attached to an acyl or alkyl group        via an ester, ether, thioether, amide, or alkyl amine linkage;    -   (vi) substitution of one or two amino acids at positions 16, 17,        20, 21, and 24 (according to the amino acid numbering of SEQ ID        NO: 1) with an amino acid selected from the group consisting of:        Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine        (Ac-Phe), wherein the amino acid of the group is covalently        attached to a hydrophilic moiety;    -   (vii) Asp at position 15 (according to the numbering of SEQ ID        NO: 1) is substituted with cysteic acid, glutamic acid,        homoglutamic acid, and homocysteic acid;    -   (viii) Ser at position 16 (according to the numbering of SEQ ID        NO: 1) is substituted with cysteic acid, glutamic acid,        homoglutamic acid, and homocysteic acid;    -   (ix) Arg at position 17 is replaced with Gln, Arg at position 18        is replaced with Ala, Asp at position 21 is replaced with Glu,        Val at position 23 is replaced with Ile, and Gln at position 24        is replaced with Ala (according to amino acid numbering of SEQ        ID NO: 1);    -   (x) Ser at position 16 is replaced with Glu, Gln at position 20        is replaced with Glu, or Gln at position 24 is replaced with        Glu(according to the amino acid numbering of SEQ ID NO: 1);

wherein C is selected from the group consisting of:

-   -   (vii) X;    -   (viii) X-Y;    -   (ix) X-Y-Z;    -   (x) X-Y-Z-R10;        wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid;        Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl        phenylalanine (Ac-Phe), or a charged amino acid; wherein R10 is        selected from a group consisting of SEQ ID NOs: 1221, 1226,        1227, and 1250.

In specific aspects in which the glucagon antagonist peptide comprisesthe general structure A-B-C, the glucagon antagonist peptide comprisesan oxy derivative of PLA. As used herein “oxy derivative of PLA” refersto a compound comprising a modified structure of PLA in which thehydroxyl group has been replaced with O—R₁₁, wherein R₁₁ is a chemicalmoiety. In this regard, the oxy derivative of PLA can be, for example,an ester of PLA or an ether of PLA.

Methods of making oxy derivatives of PLA are known in the art. Forexample, when the oxy derivative is an ester of PLA, the ester may beformed by upon reaction of the hydroxyl of PLA with a carbonyl bearing anucleophile. The nucleophile can be any suitable nucleophile, including,but not limited to an amine or hydroxyl. Accordingly, the ester of PLAcan comprise the structure of Formula V:

wherein R7 is an ester formed upon reaction of the hydroxyl of PLA witha carbonyl bearing a nucleophile.

The carbonyl bearing a nucleophile (which reacts with the hydroxyl ofPLA to form an ester) can be, for example, a carboxylic acid, acarboxylic acid derivative, or an activated ester of a carboxylic acid.The carboxylic acid derivative can be, but is not limited to, an acylchloride, an acid anhydride, an amide, an ester, or a nitrile. Theactivated ester of a carboxylic acid can be, for example,N-hydroxysuccinimide (NHS), tosylate (Tos), a carbodiimide, or ahexafluorophosphate. In some embodiments, the carbodiimide is1,3-dicyclohexylcarbodiimide (DCC), 1,1′-carbonyldiimidazole (CDI),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or1,3-diisopropylcarbodiimide (DICD). In some embodiments, thehexafluorophosphate is selected from a group consisting ofhexafluorophosphate benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU), ando-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU).

Methods of making ethers from reaction with a hydroxyl group (e.g., thehydroxyl of PLA) also are known in the art. For example, the hydroxylgroup of PLA may be reacted with a halogenated alkyl or tosylated alkylalcohol to form an ether bond.

Generally, the chemical moiety of R₁₁ is one which does not decrease theactivity of the glucagon antagonist peptide. In some embodiments, thechemical moiety enhances the activity, stability, and/or solubility ofthe glucagon antagonist peptide.

In some embodiments, the chemical moiety bound to PLA via anoxygen-containing bond (e.g., via an ester or ether bond) is a polymer(e.g., a polyalkylene glycol), a carbohydrate, an amino acid, a peptide,or a lipid, e.g., a fatty acid or a steroid.

In specific embodiments, the chemical moiety is an amino acid, which,optionally, is a part of a peptide, such that Formula V is adepsipeptide. In this regard, PLA may be at a position other than theN-terminal amino acid residue of the glucagon antagonist peptide, suchthat the glucagon antagonist peptide comprises one or more (e.g., 1, 2,3, 4, 5, 6, or more) amino acids N-terminal to the PLA residue. Forexample, the glucagon antagonist peptide can comprise PLA at position n,wherein n is 2, 3, 4, 5, or 6 of the glucagon antagonist peptide.

The amino acids N-terminal to the PLA residue may be synthetic ornaturally-occurring. In specific aspects, the amino acids which areN-terminal to PLA are naturally-occurring amino acids. In someembodiments, the amino acids which are N-terminal to PLA are theN-terminal amino acids of native glucagon. For example, the glucagonantagonist peptide can comprise at the N-terminus the amino acidsequence of any of SEQ ID NOs: 1154-1158, wherein PLA is linked tothreonine via an ester bond:

SEQ ID NO: 1154 His-Ser-Gln-Gly-Thr-PLA SEQ ID NO: 1155Ser-Gln-Gly-Thr-PLA SEQ ID NO: 1156 Gln-Gly-Thr-PLA SEQ ID NO: 1157Gly-Thr-PLA SEQ ID NO: 1158 Thr-PLA

In alternative embodiments, one or more of the N-terminal amino acidsmay be substituted with an amino acid other than the amino acid ofnative glucagon. For example, when the glucagon antagonist comprises PLAas the amino acid at position 5 or 6, the amino acid at position 1and/or position 2 may be an amino acid which reduces susceptibility tocleavage by dipeptidyl peptidase IV. More particularly, in someembodiments, position 1 of the glucagon antagonist peptide is an aminoacid selected from the group consisting of D-histidine, alpha,alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine,alpha-methyl histidine, imidazole acetic acid, desaminohistidine,hydroxyl-histidine, acetyl-histidine and homo-histidine. Moreparticularly, in some embodiments, position 2 of the glucagon antagonistpeptide is an amino acid selected from the group consisting of D-serine,D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, andaminoisobutyric acid (AIB). Also, for example, when the glucagonantagonist peptide comprises PLA as the amino acid at position 4, 5, or6, the amino acid at position 3 of the glucagon antagonist peptide maybe glutamic acid, as opposed to the native glutamine residue of nativeglucagon. In an exemplary embodiment of the present disclosures, theglucagon antagonist comprises at the N-terminus the amino acid sequenceof any of SEQ ID NOs: 1159-1161.

With respect to the glucagon antagonist peptides comprising a compoundof Formula V, the polymer which is the chemical moiety bound to PLA maybe any polymer, provided that it can react with the hydroxyl group ofPLA. The polymer may be one that naturally or normally comprises acarbonyl bearing a nucleophile. Alternatively, the polymer may be onewhich was derivatized to comprise the carbonyl bearing the carbonyl. Thepolymer may be a derivatized polymer of any of: polyamides,polycarbonates, polyalkylenes and derivatives thereof including,polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,polymers of acrylic and methacrylic esters, including poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate), polyvinyl polymers includingpolyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinylhalides, poly(vinyl acetate), and polyvinylpyrrolidone, polyglycolides,polysiloxanes, polyurethanes and co-polymers thereof, cellulosesincluding alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, celluloseacetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, and cellulose sulphate sodium salt, polypropylene,polyethylenes including poly(ethylene glycol), poly(ethylene oxide), andpoly(ethylene terephthalate), and polystyrene.

The polymer can be a biodegradable polymer, including a syntheticbiodegradable polymer (e.g., polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid),poly(valeric acid), and poly(lactide-cocaprolactone)), and a naturalbiodegradable polymer (e.g., alginate and other polysaccharidesincluding dextran and cellulose, collagen, chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), albumin and other hydrophilicproteins (e.g., zein and other prolamines and hydrophobic proteins)), aswell as any copolymer or mixture thereof. In general, these materialsdegrade either by enzymatic hydrolysis or exposure to water in vivo, bysurface or bulk erosion.

The polymer can be a bioadhesive polymer, such as a bioerodible hydrogeldescribed by H. S. Sawhney, C. P. Pathak and J. A. Hubbell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some aspects, the polymer is a water-soluble polymer. Suitablewater-soluble polymers are known in the art and include, for example,polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel),hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose,hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose,hydroxypropyl pentylcellulose, methyl cellulose, ethylcellulose(Ethocel), hydroxyethyl cellulose, various alkyl celluloses andhydroxyalkyl celluloses, various cellulose ethers, cellulose acetate,carboxymethyl cellulose, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, vinyl acetate/crotonic acid copolymers,poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylicacid copolymers, polymethacrylic acid, polymethylmethacrylate, maleicanhydride/methyl vinyl ether copolymers, poly vinyl alcohol, sodium andcalcium polyacrylic acid, polyacrylic acid, acidic carboxy polymers,carboxypolymethylene, carboxyvinyl polymers, polyoxyethylenepolyoxypropylene copolymer, polymethylvinylether co-maleic anhydride,carboxymethylamide, potassium methacrylate divinylbenzene co-polymer,polyoxyethyleneglycols, polyethylene oxide, and derivatives, salts, andcombinations thereof.

In specific embodiments, the polymer is a polyalkylene glycol,including, for example, polyethylene glycol (PEG).

In aspects in which A (of A-B-C) is an oxyderivative of PLA, thechemical moiety bound to PLA is a carbohydrate. The carbohydrate may beany carbohydrate provided that it comprises or is made to comprise acarbonyl with an alpha leaving group. The carbohydrate, for example, maybe one which has been derivatized to comprise a carbonyl with an alphaleaving group. In this regard, the carbohydrate may be a derivatizedform of a monosaccharide (e.g., glucose, galactose, fructose), adisaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide(e.g., raffinose, stachyose), a polysaccharide (a starch, amylase,amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan,fucoidan, galactomannan.

In aspects in which A (of A-B-C) is an oxyderivative of PLA, thechemical moiety bound to PLA can be a lipid. The lipid may be any lipidcomprising a carbonyl with an alpha leaving group. The lipid, forexample, may be one which is derivatized to comprise the carbonyl. Inthis regard, the lipid, may be a derivative of a fatty acid (e.g., aC4-C30 fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane,N-acyl ethanolamine), glycerolipid (e.g., mono-, di-, tri-substitutedglycerols), glycerophospholipid (e.g., phosphatidylcholine,phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine),sphingolipid (e.g., sphingosine, ceramide), sterol lipid (e.g., steroid,cholesterol), prenol lipid, saccharolipid, or a polyketide oil, wax,cholesterol, sterol, fat-soluble vitamin, monoglyceride, diglyceride,triglyceride, a phospholipid.

In some embodiments, R7 has a molecular weight of about 100 kDa or less,e.g., about 90 kDa or less, about 80 kDa or less, about 70 kDa or less,about 60 kDa or less, about 50 kDa or less, about 40 kDa or less.Accordingly, R7 can have a molecular weight of about 35 kDa or less,about 30 kDa or less, about 25 kDa or less, about 20 kDa or less, about15 kDa or less, about 10 kDa or less, about 5 kDa or less, or about 1kDa.

In alternative embodiments, the glucagon antagonist peptide of structureA-B-C comprises, as A, a peptide of 2 to 6 amino acids in which twoconsecutive amino acids of the peptide are linked via an ester or etherbond. The ester or ether bond may be, e.g., between amino acids 2 and 3,3 and 4, 4 and 5, or 5 and 6. Optionally the peptide may be furthermodified by covalent linkage to another chemical moiety includinglinkage to a polymer (e.g. a hydrophilic polymer), alkylation, oracylation.

The peptide may comprise any amino acids, synthetic or naturallyoccurring, provided that at least two consecutive amino acids of thepeptide are linked via an ester or ether bond. In a specific embodiment,the peptide comprises amino acids of native glucagon. For example, thepeptide can comprise j to 6 of native glucagon (SEQ ID NO: 1), wherein jis 1, 2, 3, 4, or 5. Alternatively, the peptide can comprise an aminoacid sequence based on the N-terminus of SEQ ID NO: 1 with one or moreamino acid modifications. The amino acid at position 1 and/or position 2may be an amino acid which reduces susceptibility to cleavage bydipeptidyl peptidase IV. For instance, the peptide can comprise atposition 1 of the glucagon antagonist peptide (glucagon antagonist) anamino acid selected from the group consisting of D-histidine, alpha,alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine,alpha-methyl histidine, imidazole acetic acid, desaminohistidine,hydroxyl-histidine, acetyl-histidine and homo-histidine. Moreparticularly, in some embodiments, position 2 of the antagonist peptideis an amino acid selected from the group consisting of D-serine,D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, andaminoisobutyric acid (AIB). Also, for example, the amino acid atposition 3 of the glucagon antagonist may be glutamic acid, as opposedto the native glutamine residue of native glucagon. Accordingly, theglucagon antagonist can comprise an amino acid sequence of:

Xaa₁-Xaa₂-Xaa₃-Thr-Gly-Phe; (SEQ ID NO: 1168) Xaa₂-Xaa₃-Thr-Gly-Phe;(SEQ ID NO: 1169) or Xaa₃-Thr-Gly-Phe; (SEQ ID NO: 1170)

wherein Xaa₁ is selected from a group consisting of: His, D-histidine,alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine,alpha-methyl histidine, imidazole acetic acid, desaminohistidine,hydroxyl-histidine, acetyl-histidine and homo-histidine; Xaa₂ isselected from a group consisting of: Ser, D-serine, D-alanine, valine,glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid(AIB); and Xaa₃ is Gln or Glu; wherein at least one bond between theamino acids of SEQ ID NO: 1168, 1169, or 1170 is an ester or ether bond.

With regard to the glucagon antagonist peptide comprising the generalstructure A-B-C, B represents amino acids of native glucagon, e.g., i to26 of SEQ ID NO: 1, wherein i is 3, 4, 5, 6, or 7, optionally comprisingone or more amino acid modifications. In a specific embodiment, Brepresents amino acids 7 to 26 of SEQ ID NO: 1, optionally furthermodified.

In some embodiments, B is modified by up to three amino acidmodifications. For example, B, which represents native amino acidsequence of SEQ ID NO: 1 is modified by one or more conservative aminoacid modifications.

In other embodiments, B comprises one or more amino acid modificationsselected from the group consisting of (iv) to (ix), as described herein.In specific embodiments, B comprises one or both of the amino acidmodifications (v) and (vi). In further specific embodiments, B comprisesone or a combination of amino acid modifications selected from the groupconsisting of (iv), (vii), (viii), and (ix), in addition to (v) and(vi). In a further specific embodiment in which the peptide comprises(1) a stabilized alpha helix through means described herein (e.g.,through an intramolecular bridge, or incorporation of one or more alpha,alpha-di-substituted amino acids, or an acidic amino acid at position 16(according to the numbering of SEQ ID NO:1), or a combination thereof;(2) a C-terminal amide or ester in place of a C-terminal carboxylate,and (3) a general structure of A-B-C, B comprises one or a combinationof amino acid modifications selected from the group consisting of (iv),(vii), (viii), (ix), and (x), in addition to (v) and (vi).

In another specific embodiment, the glucagon antagonist peptidecomprises one or more charged amino acids at the C-terminus. Forexample, Y and/or Z can be a charged amino acid, e.g., Lys, Arg, His,Asp, and Glu. In yet another embodiment, the glucagon antagonist peptidecomprises one to two charged amino acids (e.g., Lys, Arg, His, Asp, andGlu) C-terminal to Z. In specific aspects, Z followed by one to twocharged amino acids does not comprise R10. In some aspects, Y is Asp.

The glucagon antagonist peptide in some embodiments comprises ahydrophilic moiety covalently bound to an amino acid residue of theglucagon antagonist, as described herein. For example, the glucagonantagonist can comprise a hydrophilic moiety covalently attached to anamino acid at position 1, 16, 20, 21, or 24 according to the numberingof SEQ ID NO: 1 or to the N- or C-terminal amino acid of the glucagonantagonist peptide. In another embodiment, the hydrophilic moiety isattached to the C-terminal amino acid of the glucagon antagonistpeptide, which in some cases, is 1 or 11 amino acids C-terminal to Z. Inyet another embodiment, the hydrophilic moiety is attached to PLA, whenA is PLA, PLA-Phe, or PLA-Thr-Phe, wherein PLA is modified to comprisethe hydrophilic moiety. In another embodiment, an amino acid comprisinga hydrophilic moiety is added to the N- or C-terminus of the glucagonantagonist.

In specific embodiments, the hydrophilic moiety is attached to a Cysresidue of the glucagon antagonist peptide comprising the generalstructure A-B-C. In this regard, the amino acid at position 16, 21, 24,or 29 (according to the numbering of native glucagon or the N- orC-terminal amino acid may be substituted with a Cys residue.Alternatively, a Cys residue comprising a hydrophilic moiety may beadded to the C-terminus of the peptide comprising the general structureA-B-C as position 30 or as position 40, e.g., when the peptidecomprising the general structure A-B-C comprises a C-terminal extension(positions according to the amino acid numbering of SEQ ID NO: 1).Alternatively, the hydrophilic moiety may be attached to the PLA of thepeptide comprising the general structure A-B-C via the hydroxyl moietyof PLA. The hydrophilic moiety can be any of those described herein,including, for example, polyethylene glycol.

In a specific aspect, the glucagon antagonist peptide comprising thegeneral structure A-B-C comprises a stabilized alpha helix by virtue ofcomprising modifications as taught herein under “Stabilization of theAlpha Helix Structure.” Accordingly, the glucagon antagonist peptide insome aspects, comprises an intramolecular bridge and/or one or morealpha, alpha di-substituted amino acids within the C-terminal portion ofthe peptide (residues 12-29 according to the numbering of SEQ ID NO: 1).In some aspects, a stabilized alpha helix is provided by incorporationof an intramolecular bridge into the glucagon antagonist peptide. In oneembodiment, the intramolecular bridge is a lactam bridge. The lactambridge may be between the amino acids at positions 9 and 12, the aminoacids at positions 12 and 16, the amino acids at positions 16 and 20,the amino acids at positions 20 and 24, or the amino acids at positions24 and 28 (according to the amino acid numbering of SEQ ID NO: 1). In aspecific embodiment, the amino acids at positions 12 and 16 or atpositions 16 and 20 (according to the amino acid numbering of SEQ IDNO: 1) are linked via a lactam bridge. Other positions of the lactambridge are contemplated.

Additionally or alternatively, the peptide comprising the generalstructure A-B-C can comprise a stabilized alpha helix by virtue ofcomprising an alpha, alpha di-substituted amino acid at, for example,any of positions 16, 20, 21, or 24 (according to the amino acidnumbering of SEQ ID NO: 1). In one embodiment, the alpha, alphadi-substituted amino acid is AIB. In a specific aspect, the AIB islocated at position 16 (according to the numbering of SEQ ID NO: 1).

Alternatively or additionally, the glucagon antagonist peptidecomprising the general structure A-B-C may be modified to comprise anacidic amino acid at position 16 (according to the numbering of SEQ IDNO: 1), which modification enhances the stability of the alpha helix.The acidic amino acid, in one embodiment, is an amino acid comprising aside chain sulfonic acid or a side chain carboxylic acid. In a morespecific embodiment, the acidic amino acid is selected from the groupconsisting of Glu, Asp, homoglutamic acid, a sulfonic acid derivative ofCys, cysteic acid, homocysteic acid, Asp, and an alkylated derivative ofCys having the structure of

wherein X₅ is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl.

In a specific embodiment, the glucagon antagonist peptide which is aglucagon antagonist/GLP-1 agonist may comprise the amino acid sequenceof any of SEQ ID NOs: 1260-1270, 1273-1278, 1280-1288, 1290-1296, 1303,1304, 1306, and 1314-1318, or comprising the amino acid sequence of anyof Peptides 2-6 of Table A, Peptides 1-8 of Table B, and Peptides 2-6,8, and 9 of Table C:

TABLE A GLP-1 Glu EC₅₀ IC₅₀ (nM) (nM) 1 E9, K12, FTSEYSKYLDERRAQDFVQWLMNTGP 1451  762 E16 SSGAPPPS 2 E9, FTSEYSKYLDERRAQDFVQWLMNTGP 63 2008 K12E16 SSGAPPPS (lactam) 3 E9, FTSEYSKYLDERRAKDFVQWLMNTGP 36   42 E16K20 SSGAPPPS (lactam) 4 D9, FTSDYSKYLDERRAQDFVQWLMNTGP 118.7  828 K12E16 SSGAPPPS (Lactam) 5 [PLA6, PLA- 6   72 E9, TSEYSKYLDERRAQDFVQWLMNTGPS K12E16 SGAPPPS (Lactam) 6[PLA6,  PLA- 20   20 E9, TSEYSKYLDERRAKDFVQWLMNTGPS E16K20 SGAPPPS(Lactam)]

TABLE B GLP-1 Glucagon EC50 IC50 (nM) (nM) Glucagon   0.2~1.0*HSQGTFTSDYSKY1DSRRAQDFVQWLMNT GLP-1 (aa 1-30)   0.02~0.1HAEGTFTSDVSSYLEGQAAKEFIAW1VKGR 1 [PLA6, D9, E16K20(lactam), D28]G(6-29) 52~5  10~30 PLA TSDYSKY1DERRAKDFVQWLMDT 2[PLA6, D9, K12E16(Lactam), D28]G(6-29) 177  63PLA TSDYSKY1DERRAQDFVQWLMDT 3[PLA6, D9, E16, K20E24(Lactam), D28]G(6-29) 239  74PLA TSDYSKYLDERRAEDFVKWLMDT 4[PLA6, D9, E16, E24K28(lactam), D28]G(6~29) 289  22PLA TSDYSKYLDERRAQDFVEWIMKT 5 [E9, E16K20(lactam), D28]G(4~29) 151 10~30 GTFTSEYSKYLDERRAKDFVQWLMDT 6 [E9, E16K20(lactam), D28]G(2~29) 203 49 (PA) SQGTFTSEYSKYIDERRAKDFVQWLMDT 7[A2E3, E16K20(Lactam),D28]G(2~29) 175  63 AEGTFTSEYSKYLDERRAKDFVQWLMDT 8[A2E3, E16K20(Lactam), D28]G(1~29)   0.2 130 (PA)HAEGTFTSEYSKYIDERRAKDFVQWLMDT 9 ANK2 (Bayer peptide)   0.28 agonistHSQGTFTSDY ARYLDARRAREFIKWL VRGRG

TABLE C Glucagon (6-CEX) Analogs 1 E9, K12, FTSEYSKYIDERRAQDFVQWIMNTGPSSGAPPPS 1451  762 E16 2 E9, K12E16FTSEYSKYIDERRAQDFVQWIMNTGPSSGAPPPS   63 2008 (lactam) 3 E9, E16K20FTSEYSKYIDERRAKDFVQWIMNTGPSSGAPPPS   36   42 (lactam) 4 D9, K12E20FTSDYSKYIDERRAQDFVQWIMNTGPSSGAPPPS   18  828 (lactam) 5 [PLA6, E9,PLA-TSEYSKYIDERRAQDFVQWLMNTGPSSGAPPPS    6   72 K12E20 (lactam) 6[PLA6, E9, PLA-TSEYSKYLDERRAKDFVQWLMNTGPSSGAPPPS   20   20 E16K20(Lactam)] Glucagon D⁹(6-29) analogs GLP-1 Glucagon EC50 IC50 (nM) (nM) 7PLA6, D9,  PLA-TSDYSKYLDSRRAQDFVQWLMDT −700 tbd D28 8 PLA6, D9,PLA-TSDYSKYLDERRAQDFVQWLMDT   21   13 Kl2E20 (Lactam) 9 PLA6, D9,PLA-TSDYSKYLDERRAKDFVQWLMDT    4    6 E16K20 (lactam)

In certain embodiments, the glucagon antagonist peptide comprising thegeneral structure A-B-C is a glucagon antagonist/GLP-1 agonist whichexhibits at least about 50% of the maximum agonism achieved by nativeGLP-1 at the GLP-1 receptor and at least about 50% inhibition of themaximum response achieved by native glucagon at the glucagon receptor.In other specific embodiments, the glucagon antagonist peptide exhibitsat least about 55%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, or about 100% of themaximum agonism achieved by native GLP-1 at the GLP-1 receptor.Alternatively or additionally, the glucagon antagonist peptide mayexhibit at least about 55%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, or about 100%inhibition of the maximum response achieved by native glucagon at theglucagon receptor.

In other embodiments, the glucagon antagonist peptide comprises an acylgroup or alkyl group as described herein. For example, the acylation oralkylation can occur off the side chain of the amino acid at position10, 20, or 24, according to the numbering of SEQ ID NO: 1. In analternative embodiment, the acylation or alkylation occurs off the sidechain of the C-terminal amino acid of the glucagon antagonist, which insome cases, is 1 or 11 amino acids C-terminal to Z. In yet anotherembodiment, when A is PLA, PLA-Phe, or PLA-Thr-Phe, the PLA is modifiedto comprise an acyl or alkyl group.

In certain embodiments of the present disclosures, the glucagonantagonist comprises the amino acid sequence of any of SEQ ID NOs: 1162,1164-1167, and 1171 or structures of any of the peptides in Tables D-L.

TABLE D Receptor cAMP Binding Inhibition Peptide IC₅₀(nM) IC₅₀(nM)Glucagon 1-2.5 N/A [Glu⁹]Glucagon(aa2-29)-NH₂ 14 partial antagonist[Glu⁹]Glucagon(aa4-29)-NH₂ 136 128 [Glu⁹]Glucagon(aa5-29)-NH₂ 37 74[Glu⁹]Glucagon(aa6-29)-NH₂ 36 97 Glu⁹ is glutamic acid at position 9according to the numbering of native glucagon.

TABLE E Receptor cAMP cAMP Binding Induction Inhibition Cmpd. # PeptideIC₅₀(nM) EC₅₀(nM) IC₅₀(nM) Glucagon  1.75-0.31 0.21 ± 0.11 N/A [desHis¹, Glu⁹]Glucagon-NH₂ 36.90 ± 0.32 65 ± 37 1862 ± 1234 [desHis¹,Glu⁹, Phe²⁵, Leu²⁷]Glucagon-NH₂ 12.59 ± 0.41 81 ± 23 N/A* 5 [desHis¹,desPhe⁶]Glucagon-NH₂ 129.55 ± 44.9  1178 ± 105  N/A* 6 [desHis¹, Leu⁴,Glu⁹]Glucagon-NH₂ 36.88 ± 0.03 318 ± 112 102 ± 52  4B [desHis¹,hCys⁹(SO₃ ⁻), Phe²⁵, Leu²⁷] 13.90 ± 0.37 430 ± 45  N/A* Glucagon-NH₂ 5B[desHis¹, desPhe⁶, hCys⁹(SO₃ ⁻), Phe²⁵, 53.32 ± 9.97 3212 ± 368  9217 ±3176 Leu²⁷]Glucagon-NH₂ 6B [desHis¹, Leu⁴, hCys⁹(SO₃ ⁻), Phe²⁵, Leu²⁷]1614 ± 1132 4456 ± 1469 Glucagon-NH₂ *not an antagonist amino acidpositions according to the numbering of native glucagon indicated bysuperscripted numbers

TABLE F peptide no. peptide residue 9 IC₅₀ (nM)^(α) Glucagon

1.50 (1.0~2.5)* 1 [desHis¹, Glu⁹]glucagon-NH₂

14.08 ± 0.34 2 [hGlu⁹]Glucagon(aa2-29)-NH₂

8.10 ± 0.40 3 [(CSA-1]⁹]Glucagon(aa2-29)- NH₂

12.66 ± 0.13 4 [(CSA-2)⁹]Glucagon(aa2-29)- NH₂

13.28 ± 0.78 5 [β-hGlu⁹]Glucagon(aa2-29)- NH₂

37.10 ± 0.34 6 [(NSG-1)⁹]Glucagon(aa2-29)- NH₂

983 ± 82 7 [(NSG-2)⁹]Glucagon(aa2-29)- NH₂

2348 ± 382 *EC50 (nM) hGlu = homoglutamic acid; amino acid positionsaccording to the numbering of native glucagon indicated by superscriptednumbers

TABLE G cAMP peptide no. peptide residue 9 IC₅₀(nM)^(a) pA₂ ^(b) (I/A)₅₀^(c) 8 [Glu⁹]Glucagon(aa4-29)- Glu  136.0 ± 17.84 7.05 ± 1.01 1375 NH₂ 9[Leu⁴, Glu⁹]Glucagon(aa4- Glu 36.38 ± 8.69 NA^(d) NA 29)-NH₂ 10[Glu⁹]Glucagon(aa5-29)- Glu 37.38 ± 3.41 6.94 ± 0.34 390 NH₂ 11[Glu⁹]Glucagon(aa6-29)- Glu 36.35 ± 5.23 7.16 ± 0.27 486 NH₂ 12[hGlu⁹]Glucagon(aa6-29)- hGlu 162.9 ± 70.8 6.27 ± 0.11 2361 NH₂ 13[(CSA-1)⁹]Glucagon(aa6- CSA-1 107.3 ± 5.37 6.68 ± 1.05 506 29)-NH₂ 14[(CSA-2)⁹]Glucagon(aa6- CSA-2 146.4 ± 36.9 6.64 ± 0.29 580 29)-NH₂ 15Glucagon(aa6-29)-NH₂ Asp 1894 ± 383 6.94 ± 0.63 1730 16[Lys⁹]Glucagon(aa6-29)- Lys  5779 ± 1382 6.58 ± 0.60 1990 NH₂ 17[Glu⁹]Glucagon(aa7-29)- Glu >10000 ND^(e) ND NH₂ amino acid positionsaccording to the numbering of native glucagon indicated by superscriptednumbers ^(a)Data are average ± STD for at least three independentexperiments. ^(b)pA₂, the negative logarithm of the concentration of theantagonist that reduce the response to 1unit of the agonist to theresponse obtained from 0.5 unit of agonist. Data are average ± STD forat least two duplicate experiments. ^(c)(I/A)₅₀, the inhibition index,the ratio of inhibitor IC₅₀ to the added constant glucagon (0.1-0.2 nM).Data are average of at least three independent experiments andnormalized by the EC₅₀ ^(d)NA, not full antagonist. ^(e)ND, notdetected.

TABLE H cAMP Peptide IC₅₀(nM) pA₂ IC₅₀(nM) Glucagon 1.0~2.5 (EC50)[desHis¹, Glu⁹]glucagon-NH₂ 14.08 ± 0.34 NA 1089 (partial antagonist)[hCys⁹(SO₃H)]Glucagon(aa2- 13.16 ± 1.0 NA 146.6 29)-NH₂ (partialantagonist) [hCys⁹(SO₃H)]Glucagon(aa4- 41.55 ± 4.79 7.22 ± 1.09 68.429)-NH₂ [hCys⁹(SO₃H)]Glucagon(aa5- 33.85 ± 9.38 6.77 ± 0.33 98.3 29)-NH₂[hCys⁹(SO₃H)]Glucagon(aa6-29)- 59.11 ± 18.10 7.16 ± 0.51 133.4 NH₂ aminoacid positions according to the numbering of native glucagon indicatedby superscripted numbers

TABLE I IC₅₀(nM) receptor cAMP Peptide Residue 9 binding pA₂ IC₅₀(nM)Glucagon Asp 1.0~2.5 0.05~0.15 (EC₅₀) [E⁹]Glucagon(aa6-29)-NH₂ Glu 36.35± 5.23 7.16 ± 0.27 97.2 [hCys(SO₃)9]Glucagon(aa6- hCys(SO₃) 59.11 ±18.10 7.16 ± 0.51 133.4 29)-NH₂ [hE⁹]Glucagon(aa6-29)-NH₂ hGlu 162.9 ±70.8 6.27 ± 0.11 472.2 [C⁹(SCH₂COOH)]Glucagon(aa6- CSA-1 107.3 ± 5.376.68 ± 1.05 101.2 29)-NH₂ [C⁹(SCH₂CH₂COOH)]Glucagon CSA-2 146.4 ± 36.96.64 ± 0.29 116 (aa6-29)-NH₂ Glucagon(aa6-29)-NH₂ Asp  1670 ± — 6.94 ±0.63 346 [K⁹]Glucagon(aa6-29)-NH₂ Lys  3236 ± — 6.58 ± 0.60 398 aminoacid positions according to the numbering of native glucagon indicatedby superscripted numbers

TABLE J IC50(nM) (cAMP, inhibit IC50(nM) Glucagon) Solubility (Receptor0.1 nM or (%, Peptide binding) 0.2 nM pH 6-8) Glucagon  1.96 ± 0.61 0.09(EC50) [PLA6, D9]Glucagon(aa6- 13.85 ± 3.22 6.90 11 29)-NH2 [PLA6,D9]Glucagon(aa6- 15.51 ± 3.86 13.20 96 29)-COOH [PLA6, E9]Glucagon(aa6-12.33 ± 2.24 2.39 42.40 11 29)-NH2 [PLA6, 14.20 ± 0.45 40.20hCys(SO3)9]Glucagon(aa6- 29)-NH2 [PLA6, D9, D28]  9.0 ± 1.24 1.32 100Glucagon(aa6-29)-NH2 [PLA6, E9]Glucagon (aa6-  40.28 ± 11.29 24.75 1629 + CEX)-NH2 amino acid positions according to the numbering of nativeglucagon indicated by superscripted numbers

TABLE K IC₅₀(nM) IC₅₀(nM) (cAMP, inhibit Peptide (Receptor binding) 0.8mM Glucagon) Glucagon 1.0-2.5 1.44 (EC₅₀₊) [PLA⁶, E⁹]Glucagon(aa6-29)-12.34 ± 0.13 64.8 ± 3.4 NH₂ [Ac-PLA⁶, E⁹]Glucagon(aa6- ND 38.1 ± 9.229)-NH₂ [PLA^(5,) E⁹]Glucagon(aa5-29)- ND  328 ± 25 NH₂ [PLA⁴,E⁹]Glucagon(aa4-29)- ND 84.4 ± 19.5 NH₂ (partial agonist) ND: notdetected. amino acid positions according to the numbering of nativeglucagon indicated by superscripted numbers

TABLE L IC50(nM) IC50(nM) (cAMP, (Receptor inhibit 0.2 mM Peptidebinding) Glucagon) [C8(20kDaPEG), E9]Glucagon(aa6-29)-  >1000 noantagonism NH2 [PLA6, C8(20kDaPEG), 303 ± 14 236 E9]Glucagon(aa6-29)-NH2[E9, C11(20kDaPEG)]Glucagon(aa6-29)-  >1000 no antagonism NH2 [PLA6, E9, 776 ± 161 664 C11(20kDaPEG)]Glucagon(aa6-29)-NH2 [E9, C24(20kDaPEG)]Glucagon(aa6-29)-  >1000 no antagonism NH2 [PLA6, E9, 90 ± 7126 C24(20kDaPEG)]Glucagon(aa6-29)-NH2 [MCA6, E9, 208 ± 57 no antagonismC24(20kDaPEG)]Glucagon(aa6-29)-NH2 [C5(1.2kDaPEG), E9]Glucagon(aa5-29)-1081 ± 268 2281 NH2 [C5(5kDaPEG), E9]Glucagon(aa5-29)-  634 ± 174 1608NH2 [C5(20kDaPEG), E9]Glucagon(aa5-29)- 331 ± 74 976 NH2[d-Cys5(20kDaPEG), E9]Glucagon(aa5- >10000 14764 29)-NH2[K5(CH2CH2S-20kDaPEG), >10000 no antagonism E9]Glucagon(aa5-29)-NH23.4kDaPEG-dimer[C5, E9]Glucagon(aa5-  435 ± 256 1343 29)-NH2 [PLA6,C8(1.2kDaPEG), 220 ± 36 no antagonism E9]Glucagon(aa6-29)-NH2 [PLA6,C8(5kDaPEG), E9]Glucagon(aa6-  948 ± 297 216 29)-NH2 [PLA6,C8(20kDaPEG), 303 ± 14 92 E9]Glucagon(aa6-29)-NH2 [PLA6, E9,C24(1.2  4.7± 0.4 18 kDaPEG)]Glucagon(aa6-29)-NH2 [PLA6, E9, 90 ± 7 126C24(20kDaPEG)]Glucagon(aa6-29)-NH2 [MCA6, E9, 208 ± 57 no antagonismC24(20kDaPEG)]Glucagon(aa6-29)-NH2 [Phe6, E9, >10000 no antagonismC24(20kDaPEG)]Glucagon(aa6-29)-NH2

In yet other embodiments, the glucagon antagonist peptide exhibits bothglucagon antagonist activity and GLP-1 agonist activity (e.g., aglucagon antagonist, GLP-1 agonist) and the glucagon antagonist peptidecomprises:

-   -   (1) modifications that confer glucagon antagonist activity,        including but not limited to:        -   (a) substitution of the Phe at position 6 with PLA            (according to amino acid numbering of wild type glucagon),            optionally with deletion of 1 to 5 amino acids from the            N-terminus of wild type glucagon; or        -   (b) deletion of 2 to 5 amino acids from the N-terminus of            wild type glucagon; optionally with substitution of Asp at            position 9 of wild type glucagon with glutamic acid,            homoglutamic acid or a sulfonic acid derivative of cysteine            (according to amino acid numbering of wild type glucagon);            and    -   (2) modifications that confer GLP-1 agonist activity, including        but not limited to:        -   (a) insertion or substitution of α,α-disubstituted amino            acid within amino acids 12-29 of wild type glucagon, e.g. at            one, two, three, four or more of positions 16, 17, 18, 19,            20, 21, 24 or 29 (according to the amino acid numbering of            wild type glucagon); or        -   (b) introduction of an intramolecular bridge within amino            acids 12-29 of wild type glucagon, e.g. a salt bridge or a            lactam bridge or another type of covalent bond; or        -   (c) substitution of the amino acid at one or more of            positions 2, 3, 17, 18, 21, 23, or 24 (according to the            amino acid numbering of native glucagon) with the            corresponding amino acid of GLP-1, e.g. Ser2 is replaced            with Ala, Gln3 is replaced with Glu, Arg17 is replaced with            Gln, Arg at position 18 is replaced with Ala, Asp at            position 21 is replaced with Glu, Val at position 23 is            replaced with Ile, and/or Gln at position 24 is replaced            with Ala; or        -   (d) other modifications that stabilize the alpha-helix            structure around amino acid positions 12-29 according to the            amino acid numbering of wild type glucagon;            and    -   (3) other modifications that enhance GLP-1 agonist activity,        e.g.        -   (a) a C-terminal amide or ester in place of a C-terminal            carboxylate;            and optionally    -   (4) one or more of the following modifications:        -   (a) covalent attachment to a hydrophilic moiety, such as            polyethylene glycol, e.g. at the N-terminus, or at position            6, 16, 17, 20, 21, 24, 29, 40 or at the C-terminal amino            acid; and/or        -   (b) acylation or alkylation; and optionally    -   (5) one or more of the following additional modifications:        -   (a) covalent linkage of amino acids, to the N-terminus, e.g.            1-5 amino acids to the N-terminus, optionally via an ester            bond to PLA at position 6 (according to the numbering of            wild type glucagon), optionally together with modifications            at position 1 or 2, e.g. as described herein, that improve            resistance to DPP-IV cleavage;        -   (b) deletion of amino acids at positions 29 and/or 28, and            optionally position 27 (according to the numbering of wild            type glucagon);        -   (c) covalent linkage of amino acids to the C-terminus;        -   (d) non-conservative substitutions, conservative            substitutions, additions or deletions while retaining            desired activity, for example, conservative substitutions at            one or more of positions 2, 5, 7, 10, 11, 12, 13, 14, 16,            17, 18, 19, 20, 21, 24, 27, 28 or 29, substitution of Tyr at            position 10 with Val or Phe, substitution of Lys at position            12 with Arg, substitution of one or more of these positions            with Ala;        -   (e) modification of the aspartic acid at position 15, for            example, by substitution with glutamic acid, homoglutamic            acid, cysteic acid or homocysteic acid, which may reduce            degradation; or modification of the serine at position 16,            for example, by substitution of threonine, AIB, glutamic            acid or with another negatively charged amino acid having a            side chain with a length of 4 atoms, or alternatively with            any one of glutamine, homoglutamic acid, or homocysteic            acid, which likewise may reduce degradation due to cleavage            of the Asp15-Ser16 bond;        -   (f) modification of the methionine at position 27, for            example, by substitution with leucine or norleucine, to            reduce oxidative degradation;        -   (g) modification of the Gln at position 20 or 24, e.g. by            substitution with Ala or AIB, to reduce degradation that            occurs through deamidation of Gln        -   (h) modification of Asp at position 21, e.g. by substitution            with Glu, to reduce degradation that occurs through            dehydration of Asp to form a cyclic succinimide intermediate            followed by isomerization to iso-aspartate;        -   (j) homodimerization or heterodimerization as described            herein; and        -   (k) combinations of the above.

It is understood that any of the modifications within the same class maybe combined together and/or modifications of different classes arecombined. For example, the modifications of (1)(a) may be combined with(2)(a) and (3); (1)(a) may be combined with (2)(b), e.g. lactam bridgeor salt bridge, and (3); (1)(a) may be combined with (2)(c) and (3);(1)(b) may be combined with (2)(a) and (3); (1)(b) may be combined with(2)(b), e.g. lactam bridge or salt bridge, and (3); (1)(b) may becombined with (2)(c) and (3); any of the foregoing may be combined with(4)(a) and/or (4)(b); and any of the foregoing may be combined with anyof (5)(a) through (5)(k).

In exemplary embodiments, the α,α-disubstituted amino acid AIB issubstituted at one, two, three or all of positions 16, 20, 21, or 24(according to the amino acid numbering of wild type glucagon).

In exemplary embodiments, the intramolecular bridge is a salt bridge.

In other exemplary embodiments, the intramolecular bridge is a covalentbond, e.g. a lactam bridge. In some embodiments, the lactam bridge isbetween the amino acids at positions 9 and 12, the amino acids atpositions 12 and 16, the amino acids at positions 16 and 20, the aminoacids at positions 20 and 24, or the amino acids at positions 24 and 28(according to the amino acid numbering of SEQ ID NO: 1).

In exemplary embodiments, acylation or alkylation is at position 6, 10,20 or 24 or the N-terminus or C-terminus (according to the amino acidnumbering of wild type glucagon) SEQ ID NO: 1).

In exemplary embodiments, modifications include:

-   -   (i) substitution of Asp at position 15 (according to the        numbering of SEQ ID NO: 1) with cysteic acid, glutamic acid,        homoglutamic acid, and homocysteic acid;    -   (ii) substitution of Ser at position 16 (according to the        numbering of SEQ ID NO: 1) with cysteic acid, glutamic acid,        homoglutamic acid, and homocysteic acid;    -   (iii) substitution of Asn at position 28 with a charged amino        acid;    -   (iv) substitution of Asn at position 28 with a charged amino        acid selected from the group consisting of Lys, Arg, His, Asp,        Glu, cysteic acid, and homocysteic acid;    -   (v) substitution at position 28 with Asn, Asp, or Glu;    -   (vi) substitution at position 28 with Asp;    -   (vii) substitution at position 28 with Glu;    -   (viii) substitution of Thr at position 29 with a charged amino        acid;    -   (ix) substitution of Thr at position 29 with a charged amino        acid selected from the group consisting of Lys, Arg, His, Asp,        Glu, cysteic acid, and homocysteic acid;    -   (x) substitution at position 29 with Asp, Glu, or Lys;    -   (xi) substitution at position 29 with Glu;    -   (xii) insertion of 1-3 charged amino acids after position 29;    -   (xiii) insertion after position 29 of Glu or Lys;    -   (xiv) insertion after position 29 of Gly-Lys or Lys-Lys; or        combinations thereof.

Additional Modifications of the Glucagon Antagonist Peptide

In some aspects of the present disclosures, the glucagon antagonistpeptide of any of the foregoing embodiments (e.g., the glucagonantagonist peptide of structure A-B-C, the glucagon antagonist peptidecomprising a deletion of amino acids 1-5 of SEQ ID NO: 1 and a PLA atposition 6 in place of Phe of SEQ ID NO: 1, the glucagon antagonistpeptide comprising a substitution at position 9 and a deletion ofN-terminal residues of SEQ ID NO: 1) comprises one or more further aminoacid modifications (as compared to SEQ ID NO: 1), such as any of theamino acid modifications taught herein within the sections entitled“Modifications which reduce degradation,” “Modifications which enhancesolubility,” “Other modifications,” “Acylation and alkylation.” Whilethese teachings are made in the context of the GIP agonist peptide, suchmodifications are applicable to the glucagon antagonist peptide of thepresent disclosures. It should be understood that the numbering of aminoacid positions of these sections are in accordance with the numbering ofSEQ ID NO: 1.

Accordingly, in some aspects, the glucagon antagonist peptide comprisesan amino acid modification which reduces degradation. In exemplaryaspects, the glucagon antagonist peptide comprises modifications whichprovide glucagon antagonist activity and further comprises one or twoamino acid modifications at position 15 and/or position 16, as describedherein with respect to the GIP agonist peptide. See, “Modifications thatreduce degradation.” Accordingly, in some aspects, the glucagonantagonist peptide comprises any of the modifications which conferglucagon antagonist activity and further comprises a substitution of theaspartic acid located at position 15 of the native glucagon peptide withan amino acid selected from the group consisting of cysteic acid,glutamic acid, homoglutamic acid and homocysteic acid.

In accordance with some embodiments, the glucagon antagonist peptidecomprises any of the modifications which confer glucagon antagonistactivity and further comprises a substitution of the serine at position16 (according to the numbering of native glucagon) with glutamic acid,cysteic acid, homo-glutamic acid, or homo-cysteic acid. In a specificembodiment, the serine at position 16 (according to the native glucagonsequence numbering) is replaced with glutamic acid. In a more specificaspect, the glucagon antagonist comprising such a modification comprisesa C-terminal carboxylate and is not amidated.

In some aspects, the glucagon antagonist peptide comprises any of themodifications which confer glucagon antagonist activity and furthercomprises a substitution of the Met at position 27 (according to thenumbering of SEQ ID NO: 1) with a Leu or norleucine to prevent oxidativedegradation of the peptide.

In some aspects, the glucagon antagonist peptide comprises any of themodifications which confer glucagon antagonist activity and furthercomprises a substitution of any of the Gln at position 20, the Asp atposition 21, or the Gln at position 24 (or a combination thereof)(positions according to the numbering of SEQ ID NO: 1) with anotheramino acid, as described herein with regard to the GIP agonist peptide.In some aspects, the amino acid at position 20 and/or 24 (according tothe numbering of SEQ ID NO: 1) is substituted with Ser, Thr, Ala, orAIB. In other embodiments, the amino acid at position 20 and/or 24(according to the numbering of SEQ ID NO: 1) is substituted with Lys,Arg, Orn, or citrulline. In some embodiments, the amino acid at position21 (according to the numbering of SEQ ID NO: 1) is substituted with Glu.

In yet other aspects of the present disclosures, the glucagon antagonistpeptide is modified for enhanced solubility. For example, the glucagonantagonist peptide can be modified in accordance with the teachingsunder “Modifications that enhance solubility” taught herein. Inexemplary embodiments, the glucagon antagonist peptide comprises one ortwo charged amino acids at positions 28 and 29 (according to thenumbering of SEQ ID NO: 1) and/or comprises additional charged aminoacids C-terminal to position 29 (according to the numbering of SEQ IDNO: 1).

The glucagon antagonist peptide in additional aspects comprises any ofthe modifications taught herein under “Other modifications.” Forexample, the glucagon antagonist peptide in some aspects comprises asubstitution of Lys at position 12 (according to the numbering of SEQ IDNO: 1) with Arg. In some aspects, the glucagon antagonist peptidecomprises a charge neutral group in place of the alpha carboxylate ofthe C-terminal residue.

In some embodiments, the glucagon antagonist peptide which exhibitsglucagon antagonist activity is acylated or alkylated in accordance withthe teachings found herein under “Acylation and alkylation.”

Any of the modifications described above which increase GLP-1 receptoragonist activity, glucagon receptor antagonist activity, peptidesolubility, and/or peptide stability can be applied individually or incombination.

Glucagon Analog Peptides

In addition to the peptide combinations comprising a GIP agonist peptideand a glucagon antagonist peptide, the present disclosures additionallyprovide any of the glucagon analog peptides described herein (e.g., theGIP agonist peptides, glucagon antagonist peptides) in free form (e.g.,not in combination with a different type of peptide, not conjugated toanother peptide), provided that they are not disclosed in any of thereferences cited herein, including any of International PatentApplication No. PCT/US2009/47447, International Patent ApplicationPublication Nos. WO2009/058662 and WO2009/058734, or U.S. PatentApplication Nos. 60/073,274; 61/078,171; 61/090,448; 61/151,349;61/187,578; 60/983,783; 60/983,766; 61/090,441. Accordingly, inexemplary embodiments, the glucagon analog peptide is a GIP agonistpeptide not in combination with (e.g., not conjugated to) a glucagonantagonist peptide. In alternative embodiments, the glucagon analogpeptide is a glucagon antagonist peptide, not in combination with (e.g.,not conjugated to) a GIP agonist peptide.

In some embodiments, the peptide of the present disclosures is either aGIP agonist peptide or a glucagon antagonist peptide, according to thedescriptions herein, is an analog of glucagon (SEQ ID NO: 1), andfurthermore is an analog of a peptide disclosed in any of the referencescited herein, including any of International Patent Application No.PCT/US2009/47447, International Patent Application Publication Nos.WO2009/058662 and WO2009/058734, or U.S. Patent Application Nos.60/073,274; 61/078,171; 61/090,448; 61/151,349; 61/187,578; 60/983,783;60/983,766; 61/090,441, in which one or more amino acid residues of theanalog cited in these references is changed in accordance with theteachings described herein. For example, the glucagon analog peptide ofthe present disclosures may be an analog of an amino acid sequence foundwithin Sequence Listing 2 or Sequence Listing 3 in which the sequencebegins with a Tyr, wherein the analog comprises an amino acidmodification which reduces GIP activity as described herein. Forexample, the analog may be identical in sequence to a sequence ofSequence Listing 2 or 3, but comprises instead of the Tyr at position 1,a small aliphatic residue, e.g., Ala, Gly, or has the amino acid(s) atposition 1 or at positions 1 and 2 deleted.

In some embodiments, in which the glucagon analog peptide is a GIPagonist peptide, the glucagon analog peptide can exhibit activity at theglucagon receptor in addition to activity at the GIP receptor (and,optionally, the GLP-1 receptor). In exemplary embodiments, the GIPagonist peptide exhibits tri-agonism at each of the glucagon, GIP, andGLP-1 receptors or co-agonism at each of the glucagon and GIP receptors.In such embodiments, the glucagon analog peptide exhibits at least orabout 0.1% activity of native glucagon at the glucagon receptor. Inexemplary embodiments, the GIP agonist peptide exhibits at least orabout 0.2%, at least or about 0.3%, at least or about 0.4%, at least orabout 0.5%, at least or about 0.6%, at least or about 0.7%, at least orabout 0.8%, at least or about 0.9%, at least or about 1%, at least orabout 5%, at least or about 10%, at least or about 20%, at least orabout 30%, at least or about 40%, at least or about 50%, at least orabout 60%, at least or about 70%, at least or about 75%, at least orabout 80%, at least or about 90%, at least or about 95%, or at least orabout 100% of the activity of native glucagon at the glucagon receptor.

In some embodiments, the EC50 of the GIP agonist peptide at the GIPreceptor is less than or about 50-fold, less than or about 40-fold, lessthan or about 30-fold, or less than or about 20-fold different (higheror lower) from its EC50 at the glucagon receptor. In some embodiments,the GIP potency of the GIP agonist peptide is less than or about 25-,20-, 15-, 10-, or 5-fold different (higher or lower) from its glucagonpotency. In some embodiments, the ratio of the EC50 of the GIP agonistpeptide at the GIP receptor divided by the EC50 of the GIP agonistpeptide at the glucagon receptor is less than about 100, 75, 60, 50, 40,30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratioof the GIP potency of the GIP agonist peptide compared to the glucagonpotency of the GIP agonist peptide is less than about 100, 75, 60, 50,40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, theratio of the EC50 of the GIP agonist peptide at the glucagon receptordivided by the EC50 of the GIP agonist peptide at the GIP receptor isless than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no lessthan 1. In some embodiments, the ratio of the glucagon potency of theGIP agonist peptide compared to the GIP potency of the GIP agonistpeptide is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5,and no less than 1.

In some embodiments in which the glucagon analog peptide is a GIPagonist peptide and the GIP agonist peptide is exhibits agonism at theGIP and GLP-1 receptors, the selectivity for the human GLP-1 receptor ofthe GIP agonist peptide is not at least 100-fold the selectivity of forthe human GIP receptor. In exemplary embodiments, the selectivity of theGIP agonist peptide for the human GLP-1 receptor versus the GIP receptoris less than 100-fold (e.g., less than or about 90-fold, less than orabout 80-fold, less than or about 70-fold, less than or about 60-fold,less than or about 50-fold, less than or about 40-fold, less than orabout 30-fold, less than or about 20-fold, less than or about 10-fold,less than or about 5-fold).

In some embodiments, the peptides described herein are glycosylated,amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclizedvia, e.g., a disulfide bridge, or converted into a salt (e.g., an acidaddition salt, a basic addition salt), and/or optionally dimerized,multimerized, or polymerized, or conjugated.

The peptides of the disclosure can be obtained by methods known in theart. Suitable methods of de novo synthesizing peptides are described in,for example, Chan et al., Fmoc Solid Phase Peptide Synthesis, OxfordUniversity Press, Oxford, United Kingdom, 2005; Peptide and Protein DrugAnalysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed.Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000;and U.S. Pat. No. 5,449,752.

Also, in the instances in which the peptides of the disclosure do notcomprise any non-coded or non-natural amino acids, the peptide can berecombinantly produced using a nucleic acid encoding the amino acidsequence of the analog using standard recombinant methods. See, forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994.

In some embodiments, the peptides of the disclosure are isolated. Insome embodiments, the peptides of the disclosure are purified. It isrecognized that “purity” is a relative term, and not to be necessarilyconstrued as absolute purity or absolute enrichment or absoluteselection. In some aspects, the purity is at least or about 50%, is atleast or about 60%, at least or about 70%, at least or about 80%, or atleast or about 90% (e.g., at least or about 91%, at least or about 92%,at least or about 93%, at least or about 94%, at least or about 95%, atleast or about 96%, at least or about 97%, at least or about 98%, atleast or about 99% or is approximately 100%.

In some embodiments, the peptides described herein are commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the peptides can be synthetic,recombinant, isolated, and/or purified.

The present disclosures further provides conjugates and pharmaceuticalcompositions comprising the peptide. The teachings under “Conjugates”and “Compositions, Pharmaceutical compositions” are applicable to theseembodiments.

The present disclosures further provides methods of using the peptideswhich are glucagon analogs and are either GIP agonist peptides orglucagon antagonist peptides. When the peptide is a GIP agonist peptide,the peptide may be used in any of the methods of treatment describedherein, including methods of treating a metabolic disorder, e.g.,diabetes, obesity, and the like. When the peptide is a glucagonantagonist, the peptide may be used in a method of treatinghypoglycemia, or in any method described in the teachings ofInternational Patent Application Publication Nos. WO2009/058734 andWO2009/058662.

Conjugates

The present disclosures further provide conjugates. In some aspects, theconjugate comprises a GIP agonist conjugated to a glucagon antagonistpeptide. In some aspects, the conjugate comprises at least one of theGIP agonist peptide and the glucagon antagonist peptide conjugated to aheterologous moiety. In some aspects, the conjugate comprises a GIPagonist conjugated to a glucagon antagonist peptide and at least one ofthe peptides is conjugated to a heterologous moiety.

The conjugation between the two peptides or between the peptide andheterologous moiety may involve covalent bonds, non-covalent bonds, orboth types of bonds. In some aspects, the covalent bonds are any of thecovalent linkages described herein (e.g., disulfide bonds, lactambridges, olefin metathesis, and the like). In some aspects, the covalentbonds are peptide bonds. In specific embodiments in which theconjugation involves peptide bonds, the conjugate may be a fusionpeptide comprising either or both of the GIP agonist peptide and theglucagon antagonist peptide and optionally a heterologous moiety, e.g.,a Fc receptor, or portion thereof.

In alternative embodiments, the GIP agonist peptide is conjugated to theglucagon antagonist peptide through non-covalent linkages, e.g.,electrostatic interactions, hydrogen bonds, van der Waals interactions,salt bridges, hydrophobic interactions, and the like.

The conjugation of the peptide to the other peptide and/or to theheterologous moiety may be indirect or direct conjugation, the former ofwhich may involve a linker or spacer. Suitable linkers and spacers areknown in the art and include, but not limited to, any of the linkers orspacers described herein under the sections “Acylation and alkylation”and “Linkages.”

Heterologous Moieties

As used herein, the term “heterologous moiety” is synonymous with theterm “conjugate moiety” and refers to any molecule (chemical orbiochemical, naturally-occurring or non-coded) which is different fromthe GIP agonist peptide or glucagon antagonist peptide to which it isattached. Exemplary conjugate moieties that can be linked to any of theanalogs described herein include but are not limited to a heterologouspeptide or polypeptide (including for example, a plasma protein), atargeting agent, an immunoglobulin or portion thereof (e.g., variableregion, CDR, or Fc region), a diagnostic label such as a radioisotope,fluorophore or enzymatic label, a polymer including water solublepolymers, or other therapeutic or diagnostic agents. In some embodimentsa conjugate is provided comprising a peptide of the peptide combinationand a plasma protein, wherein the plasma protein is selected from thegroup consisting of albumin, transferin, fibrinogen and globulins. Insome embodiments the plasma protein moiety of the conjugate is albuminor transferin. The conjugate in some embodiments comprises one or moreof the peptides of the peptide combinations described herein and one ormore of: a peptide (which is distinct from the GIP agonist peptide andglucagon antagonist peptide described herein), a polypeptide, a nucleicacid molecule, an antibody or fragment thereof, a polymer, a quantumdot, a small molecule, a toxin, a diagnostic agent, a carbohydrate, anamino acid.

In some embodiments, the heterologous moiety is a peptide which isdistinct from the GIP agonist peptide and glucagon antagonist peptidedescribed herein and the conjugate is a fusion peptide or a chimericpeptide. In some embodiments, the heterologous moiety is a peptideextension of 1-21 amino acids. In specific embodiments, the extension isattached to the C-terminus of the glucagon analog, e.g., to amino acidat position 29. In some embodiments, the extension comprises an aminoacid sequence of SEQ ID NO: 3 (GPSSGAPPPS), SEQ ID NO: 4 (GGPSSGAPPPS),SEQ ID NO: 8 (KRNRNNIA), or SEQ ID NO: 9 (KRNR). In specific aspects,the amino acid sequence is attached through the C-terminal amino acid ofthe peptide, e.g., amino acid at position 29. In some embodiments, theamino acid sequence of SEQ ID NOs: 3, 4, 8, and 9 is bound to amino acid29 of the peptide through a peptide bond. In some specific embodiments,the amino acid at position 29 of the glucagon analog is a Gly and theGly is fused to one of the amino acid sequences of SEQ ID NOs: 3, 4, 8,and 9.

In some embodiments, the heterologous moiety is a polymer. In someembodiments, the polymer is selected from the group consisting of:polyamides, polycarbonates, polyalkylenes and derivatives thereofincluding, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polymers of acrylic and methacrylic esters, includingpoly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

In some aspects, the polymer is a biodegradable polymer, including asynthetic biodegradable polymer (e.g., polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)),and a natural biodegradable polymer (e.g., alginate and otherpolysaccharides including dextran and cellulose, collagen, chemicalderivatives thereof. (substitutions, additions of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), albumin andother hydrophilic proteins (e.g., zein and other prolamines andhydrophobic proteins)), as well as any copolymer or mixture thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

In some aspects, the polymer is a bioadhesive polymer, such as abioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A.Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer or ahydrophilic polymer. Hydrophilic polymers are further described hereinunder “Hydrophilic Moieties.” Suitable water-soluble polymers are knownin the art and include, for example, polyvinylpyrrolidone, hydroxypropylcellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel),nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropylbutylcellulose, hydroxypropyl pentylcellulose, methyl cellulose,ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkylcelluloses and hydroxyalkyl celluloses, various cellulose ethers,cellulose acetate, carboxymethyl cellulose, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acidcopolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate,methacrylic acid copolymers, polymethacrylic acid,polymethylmethacrylate, maleic anhydride/methyl vinyl ether copolymers,poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylicacid, acidic carboxy polymers, carboxypolymethylene, carboxyvinylpolymers, polyoxyethylene polyoxypropylene copolymer,polymethylvinylether co-maleic anhydride, carboxymethylamide, potassiummethacrylate divinylbenzene co-polymer, polyoxyethyleneglycols,polyethylene oxide, and derivatives, salts, and combinations thereof.

In specific embodiments, the polymer is a polyalkylene glycol,including, for example, polyethylene glycol (PEG).

In some embodiments, the heterologous moiety is a carbohydrate. In someembodiments, the carbohydrate is a monosaccharide (e.g., glucose,galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose),an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (astarch, amylase, amylopectin, cellulose, chitin, callose, laminarin,xylan, mannan, fucoidan, galactomannan.

In some embodiments, the heterologous moiety is a lipid. The lipid, insome embodiments, is a fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin,monoglyceride, diglyceride, triglyceride, a phospholipid.

Fc Fusions

As noted above, in some embodiments, the peptides are conjugated, e.g.,fused to an immunoglobulin or portion thereof (e.g., variable region,CDR, or Fc region). Known types of immunoglobulins (Ig) include IgG,IgA, IgE, IgD or IgM. The Fc region is a C-terminal region of an Igheavy chain, which is responsible for binding to Fc receptors that carryout activities such as recycling (which results in prolonged half-life),antibody dependent cell-mediated cytotoxicity (ADCC), and complementdependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In a relatedembodiments, the Fc region may comprise one or more native or modifiedconstant regions from an immunoglobulin heavy chain, other than CH1, forexample, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for ADCC and CDC. Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(lower hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop (Sondermann etal., Nature 406: 267-273, 2000). The lower hinge region of IgE has alsobeen implicated in the FcRI binding (Henry, et al., Biochemistry 36,15568-15578, 1997). Residues involved in IgA receptor binding aredescribed in Lewis et al., (J. Immunol. 175:6694-701, 2005). Amino acidresidues involved in IgE receptor binding are described in Sayers et al.(J Biol. Chem. 279(34):35320-5, 2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγRs(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Hydrophilic Moieties

The GIP agonist peptide and/or glucagon antagonist peptide describedherein can be further modified to improve its solubility and stabilityin aqueous solutions at physiological pH, while retaining the highbiological activity relative to native glucagon. Hydrophilic moietiessuch as PEG groups can be attached to the analogs under any suitableconditions used to react a protein with an activated polymer molecule.Any means known in the art can be used, including via acylation,reductive alkylation, Michael addition, thiol alkylation or otherchemoselective conjugation/ligation methods through a reactive group onthe PEG moiety (e.g., an aldehyde, amino, ester, thiol, α-haloacetyl,maleimido or hydrazino group) to a reactive group on the target compound(e.g., an aldehyde, amino, ester, thiol, α-haloacetyl, maleimido orhydrazino group). Activating groups which can be used to link the watersoluble polymer to one or more proteins include without limitationsulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine,oxirane, 5-pyridyl, and alpha-halogenated acyl group (e.g., alpha-iodoacetic acid, alpha-bromoacetic acid, alpha-chloroacetic acid). Ifattached to the analog by reductive alkylation, the polymer selectedshould have a single reactive aldehyde so that the degree ofpolymerization is controlled. See, for example, Kinstler et al., Adv.Drug. Delivery Rev. 54: 477-485 (2002); Roberts et al., Adv. DrugDelivery Rev. 54: 459-476 (2002); and Zalipsky et al., Adv. DrugDelivery Rev. 16: 157-182 (1995).

In specific aspects, an amino acid residue of the analog having a thiolis modified with a hydrophilic moiety such as PEG. In some embodiments,the thiol is modified with maleimide-activated PEG in a Michael additionreaction to result in a PEGylated analog comprising the thioetherlinkage shown below:

In some embodiments, the thiol is modified with a haloacetyl-activatedPEG in a nucleophilic substitution reaction to result in a PEGylatedanalog comprising the thioether linkage shown below:

Suitable hydrophilic moieties include polyethylene glycol (PEG),polypropylene glycol, polyoxyethylated polyols (e.g., POG),polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde,copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethyleneglycol, mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly (.beta.-amino acids) (either homopolymers orrandom copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers (PPG) and other polyakylene oxides,polypropylene oxide/ethylene oxide copolymers, colonic acids or otherpolysaccharide polymers, Ficoll or dextran and mixtures thereof.Dextrans are polysaccharide polymers of glucose subunits, predominantlylinked by α1-6 linkages. Dextran is available in many molecular weightranges, e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD. Linear or branchedpolymers are contemplated. Resulting preparations of conjugates may beessentially monodisperse or polydisperse, and may have about 0.5, 0.7,1, 1.2, 1.5 or 2 polymer moieties per analog.

In some embodiments, the peptide of the conjugate is conjugated to ahydrophilic moiety via covalent linkage between a side chain of an aminoacid of the glucagon analog and the hydrophilic moiety. In someembodiments, the glucagon analog is conjugated to a hydrophilic moietyvia the side chain of an amino acid at position 16, 17, 21, 24, or 29, aposition within a C-terminal extension, or the C-terminal amino acid, ora combination of these positions. In some aspects, the amino acidcovalently linked to a hydrophilic moiety (e.g., the amino acidcomprising a hydrophilic moiety) is a Cys, Lys, Orn, homo-Cys, orAc-Phe, and the side chain of the amino acid is covalently bonded to ahydrophilic moiety (e.g., PEG).

rPEG

In some embodiments, the conjugate of the present disclosures comprisesthe peptide fused to an accessory analog which is capable of forming anextended conformation similar to chemical PEG (e.g., a recombinant PEG(rPEG) molecule), such as those described in International PatentApplication Publication No. WO2009/023270 and U.S. Patent ApplicationPublication No. US20080286808. The rPEG molecule in some aspects is apolypeptide comprising one or more of glycine, serine, glutamic acid,aspartic acid, alanine, or proline. In some aspects, the rPEG is ahomopolymer, e.g., poly-glycine, poly-serine, poly-glutamic acid,poly-aspartic acid, poly-alanine, or poly-proline. In other embodiments,the rPEG comprises two types of amino acids repeated, e.g.,poly(Gly-Ser), poly(Gly-Glu), poly(Gly-Ala), poly(Gly-Asp),poly(Gly-Pro), poly(Ser-Glu), etc. In some aspects, the rPEG comprisesthree different types of amino acids, e.g., poly(Gly-Ser-Glu). Inspecific aspects, the rPEG increases the half-life of the Glucagonand/or GLP-1 agonist analog. In some aspects, the rPEG comprises a netpositive or net negative charge. The rPEG in some aspects lackssecondary structure. In some embodiments, the rPEG is greater than orequal to 10 amino acids in length and in some embodiments is about 40 toabout 50 amino acids in length. The accessory peptide in some aspects isfused to the N- or C-terminus of the analog of the present disclosurethrough a peptide bond or a proteinase cleavage site, or is insertedinto the loops of the analog of the present disclosure. The rPEG in someaspects comprises an affinity tag or is linked to a PEG that is greaterthan 5 kDa. In some embodiments, the rPEG confers the analog of thepresent disclosure with an increased hydrodynamic radius, serumhalf-life, protease resistance, or solubility and in some aspectsconfers the analog with decreased immunogenicity.

Multimers

In some embodiments in which the GIP agonist peptide is conjugated tothe glucagon antagonist peptide, and the conjugate is not a fusionpeptide, the conjugate is a multimer or dimer comprising the peptides ofthe peptide combinations. The conjugate may be a hetero-multimer orheterodimer comprising the GIP agonist peptide is conjugated to theglucagon antagonist peptide. In certain embodiments, the linkerconnecting the two (or more) peptides is PEG, e.g., a 5 kDa PEG, 20 kDaPEG. In some embodiments, the linker is a disulfide bond. For example,each monomer of the dimer may comprise a Cys residue (e.g., a terminalor internally positioned Cys) and the sulfur atom of each Cys residueparticipates in the formation of the disulfide bond. In some aspects,the monomers are connected via terminal amino acids (e.g., N-terminal orC-terminal), via internal amino acids, or via a terminal amino acid ofat least one monomer and an internal amino acid of at least one othermonomer. In specific aspects, the monomers are not connected via anN-terminal amino acid. In some aspects, the monomers of the multimer areattached together in a “tail-to-tail” orientation in which theC-terminal amino acids of each monomer are attached together.

Linkages

The following two sections on linkages provide description for linking apeptide to a heterologous moiety or for dimer or multimer formation. Theskilled artisan will recognize that the teachings of one type ofconjugate may be applicable to the other type.

Linkages—Peptide to Heterologous Moieties

The conjugation of the conjugate in some embodiments is linked toconjugate moieties via direct covalent linkage by reacting targetedamino acid residues of the analog with an organic derivatizing agentthat is capable of reacting with selected side chains or the N- orC-terminal residues of these targeted amino acids. Reactive groups onthe analog or conjugate moiety include, e.g., an aldehyde, amino, ester,thiol, α-haloacetyl, maleimido or hydrazino group. Derivatizing agentsinclude, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride or other agentsknown in the art. Alternatively, the conjugate moieties can be linked tothe analog indirectly through intermediate carriers, such aspolysaccharide or polypeptide carriers. Examples of polysaccharidecarriers include aminodextran. Examples of suitable polypeptide carriersinclude polylysine, polyglutamic acid, polyaspartic acid, co-polymersthereof, and mixed polymers of these amino acids and others, e.g.,serines, to confer desirable solubility properties on the resultantloaded carrier.

Cysteinyl residues are most commonly reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid, chloroacetamide togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,alpha-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),deamidation of asparagine or glutamine, acetylation of the N-terminalamine, and/or amidation or esterification of the C-terminal carboxylicacid group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the analog. Sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of tyrosine, or tryptophan, or (f) theamide group of glutamine. These methods are described in WO87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

In some embodiments, the peptide is conjugated to a heterologous moietyvia covalent linkage between a side chain of an amino acid of theglucagon analog and the heterologous moiety. In some embodiments, theglucagon analog is conjugated to a heterologous moiety via the sidechain of an amino acid at position 16, 17, 21, 24, or 29, a positionwithin a C-terminal extension, or the C-terminal amino acid, or acombination of these positions. In some aspects, the amino acidcovalently linked to a heterologous moiety (e.g., the amino acidcomprising a heterologous moiety) is a Cys, Lys, Orn, homo-Cys, orAc-Phe, and the side chain of the amino acid is covalently bonded to aheterologous moiety.

In some embodiments, the conjugate comprises a linker that joins theglucagon analog to the heterologous moiety. In some aspects, the linkercomprises a chain of atoms from 1 to about 60, or 1 to 30 atoms orlonger, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or to 20 atoms long.In some embodiments, the chain atoms are all carbon atoms. In someembodiments, the chain atoms in the backbone of the linker are selectedfrom the group consisting of C, O, N, and S. Chain atoms and linkers maybe selected according to their expected solubility (hydrophilicity) soas to provide a more soluble conjugate. In some embodiments, the linkerprovides a functional group that is subject to cleavage by an enzyme orother catalyst or hydrolytic conditions found in the target tissue ororgan or cell. In some embodiments, the length of the linker is longenough to reduce the potential for steric hindrance. If the linker is acovalent bond or a peptidyl bond and the conjugate is a polypeptide, theentire conjugate can be a fusion protein. Such peptidyl linkers may beany length. Exemplary linkers are from about 1 to 50 amino acids inlength, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids inlength. Such fusion proteins may alternatively be produced byrecombinant genetic engineering methods known to one of ordinary skillin the art.

In some embodiments, the heterologous moiety is attached vianon-covalent or covalent bonding to the analog of the presentdisclosure. In certain aspects, the heterologous moiety is attached tothe analog of the present disclosure via a linker. Linkage can beaccomplished by covalent chemical bonds, physical forces suchelectrostatic, hydrogen, ionic, van der Waals, or hydrophobic orhydrophilic interactions. A variety of non-covalent coupling systems maybe used, including biotin-avidin, ligand/receptor, enzyme/substrate,nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,cellular adhesion molecule partners; or any binding partners orfragments thereof which have affinity for each other.

Linkages—Dimers and Multimers

In some embodiments, the two peptides of the conjugate are linkedtogether using standard linking agents and procedures known to thoseskilled in the art. For example, in some aspects, the two peptides ofthe conjugate are fused through one or more peptide bonds, with orwithout a spacer, e.g., a peptide or amino acid spacer. In suchinstances, the two peptides of the conjugate are considered as a fusionpeptide. In alternative embodiments, the two peptides of the conjugateare linked together through chemical conjugation. In some embodiments,the two peptides of the conjugate are chemically conjugated togetherthrough a linking moiety. The linking moiety in some aspects aredirectly conjugated to each peptide, or, in alternative aspects, areindirectly conjugated to each peptide through a spacer.

In some aspects, the GIP agonist peptide and glucagon antagonist peptideof the conjugate are linked together in a “tail-to-tail” orientation inwhich the C-terminal amino acids of the peptides are conjugatedtogether. In some aspects, the GIP agonist peptide and glucagonantagonist peptide of the conjugate are linked together via the sidechains of internal amino acids on each peptide. In some aspects, the GIPagonist peptide and glucagon antagonist peptide of the conjugate arelinked together via a C-terminal amino acid of one peptide and aninternal amino acid of another peptide.

In some embodiments, two peptides of the conjugate are directly linkedtogether and do not comprise a linking moiety. In some embodiments, thetwo peptides of the conjugate are linked together by conjugating both ofthe peptides to a single linking moiety that comprises at least tworeactive groups, e.g., a bifunctional linker, a bifunctional spacer. Insome embodiments, the two peptides of the conjugate are linked togetherby indirectly conjugating one or both of the peptides to the singlelinking moiety through a spacer.

In some embodiments, the C-terminal of one or more the GIP agonistpeptide and glucagon antagonist peptide of the conjugate is modified tocomprise a natural or nonnatural amino acid with a nucleophilic sidechain, such as an amino acid represented by Formula I, Formula II, orFormula III, as previously described herein (see Acylation andalkylation). In exemplary embodiments, the C-terminal amino acid of oneor more of the GIP agonist peptide and glucagon antagonist peptide isselected from the group consisting of lysine, ornithine, serine,cysteine, and homocysteine. In some embodiments, the C-terminal aminoacid of one or more peptides of the conjugate is modified to comprise anatural or nonnatural amino acid with an electrophilic side chain suchas, for example, Asp and Glu. In some embodiments, the C-terminal aminoacids of both peptides of the conjugate comprise side chains that arenulceophilic. In some embodiments, the C-terminal amino acids of bothpeptides comprise side chains that are electrophilic. In someembodiments, the C-terminal amino acid of one peptide of the conjugatecomprises a side chain that is nulceophilic, and the C-terminal aminoacid of the other peptide of the conjugate comprises a side chain thatis electrophilic.

In some embodiments, the two peptides of the conjugate are linkedtogether by directly conjugating the C-terminal amino acids of thepeptides to one another with a linking moiety. In some embodiments, thetwo peptides of the conjugate are linked by directly conjugating theC-terminal amino acid side chain of one peptide to the C-terminal aminoacid side chain of the other peptide. In some of these embodiments, theC-terminal amino acid of one peptide comprises a nucleophilic side chainand the C-terminal amino acid of the other peptide comprises anelectrophilic side chain. In some of these embodiments, both C-terminalamino acids comprise thiol side chains and linkage occurs through adisulfide bond. In some embodiments, two peptides of the conjugate arelinked through a nucleophilic side chain of the C-terminal amino acid ofone peptide to the alpha-carboxyl group of the C-terminal amino acid onthe other peptide.

In some embodiments, the two peptides of the conjugate are linkedtogether by conjugating the C-terminal amino acid side chains of both ofthe peptides to a linking moiety that comprises at least two reactivegroups before conjugation to the peptides. In some embodiments, thelinking moiety is a bifunctional linker and comprises only two reactivegroups before conjugation to the peptides. In embodiments where the twopeptides of the composition both have C-terminal amino acids withelectrophilic side chains, the linking moiety comprises two of the sameor two different nucleophilic groups (e.g. amine, hydroxyl, thiol)before conjugation to the peptides. In embodiments where the twopeptides of the composition both have C-terminal amino acids withnucleophilic side chains, the linking moiety comprises two of the sameor two different electrophilic groups (e.g. carboxyl group, activatedform of a carboxyl group, compound with a leaving group) beforeconjugation to the peptides. In embodiments where one peptide of thecomposition has a C-terminal amino acid with a nucleophilic side chainand the other peptide of the composition has a C-terminal amino acidwith an electrophilic side chain, the linking moiety comprises onenucleophilic group and one electrophilic group before conjugation to thepeptides. In some embodiments where one or more of the two peptides ofthe composition are conjugated to each other through their C-terminalalpha-carboxyl groups, the linking moiety comprising two nucleophilicgroups before conjugation to the peptides.

Composition of the Linking Moiety

The linking moiety can be any molecule with at least two reactive groups(before conjugation to the peptides) capable of reacting with thepeptides of the composition. In some embodiments the linking moiety hasonly two reactive groups and is bifunctional. The linking moiety (beforeconjugation to the peptides) can be represented by Formula VI:

wherein X and Y are independently nucleophilic or electrophilic reactivegroups. In some embodiments X and Y are either both nucleophilic groupsor both electrophilic groups. In some embodiments one of X and Y is anucleophilic group and the other of X and Y is an electrophilic group.Nonlimiting combinations of X and Y are shown below.

Both Nucleophilic Both Electrophilic Nucleophilic/Electrophilic X Y X YX Y amino amino carboxyl carboxyl amino carboxyl amino thiol carboxylacyl chloride amino acyl chloride amino hydroxyl carboxyl anhydrideamino anhydride thiol amino carboxyl Ester amino ester thiol thiolcarboxyl NHS amino NHS thiol hydroxyl carboxyl Halogen amino halogenhydroxyl amino carboxyl sulfonate ester amino sulfonate ester hydroxylthiol carboxyl maleimido amino maleimido hydroxyl hydroxyl carboxylhaloacetyl amino haloacetyl carboxyl isocyanate amino isocyanate acylchloride carboxyl thiol carboxyl acyl chloride acyl chloride thiol acylchloride acyl chloride anhydride thiol anhydride acyl chloride Esterthiol ester acyl chloride NHS thiol NHS acyl chloride Halogen thiolhalogen acyl chloride sulfonate ester thiol sulfonate ester acylchloride maleimido thiol maleimido acyl chloride haloacetyl thiolhaloacetyl acyl chloride isocyanate thiol isocyanate anhydride carboxylhydroxyl carboxyl anhydride acyl chloride hydroxyl acyl chlorideanhydride anhydride hydroxyl anhydride anhydride Ester hydroxyl esteranhydride NHS hydroxyl NHS anhydride Halogen hydroxyl halogen anhydridesulfonate ester hydroxyl sulfonate ester anhydride maleimido hydroxylmaleimido anhydride haloacetyl hydroxyl haloacetyl anhydride isocyanatehydroxyl isocyanate ester carboxyl ester acyl chloride ester anhydrideester Ester ester NHS ester Halogen ester sulfonate ester estermaleimido ester haloacetyl ester isocyanate NHS carboxyl NHS acylchloride NHS anhydride NHS Ester NHS NHS NHS Halogen NHS sulfonate esterNHS maleimido NHS haloacetyl NHS isocyanate halogen carboxyl halogenacyl chloride halogen anhydride halogen Ester halogen NHS halogenHalogen halogen sulfonate ester halogen maleimido halogen haloacetylhalogen isocyanate sulfonate ester carboxyl sulfonate ester acylchloride sulfonate ester anhydride sulfonate ester Ester sulfonate esterNHS sulfonate ester Halogen sulfonate ester sulfonate ester sulfonateester maleimido sulfonate ester haloacetyl sulfonate ester isocyanatemaleimido carboxyl maleimido acyl chloride maleimido anhydride maleimidoEster maleimido NHS maleimido Halogen maleimido sulfonate estermaleimido maleimido maleimido haloacetyl maleimido isocyanate haloacetylcarboxyl haloacetyl acyl chloride haloacetyl anhydride haloacetyl Esterhaloacetyl NHS haloacetyl Halogen haloacetyl sulfonate ester haloacetylmaleimido haloacetyl haloacetyl haloacetyl isocyanate isocyanatecarboxyl isocyanate acyl chloride isocyanate anhydride isocyanate Esterisocyanate NHS isocyanate Halogen isocyanate sulfonate ester isocyanatemaleimido isocyanate haloacetyl isocyanate isocyanateOther nonlimiting examples of reactive groups include pyridyldithiol,aryl azide, diazirine, carbodiimide, and hydrazide. In some embodimentsat least one reactive group of the linking moiety before conjugation tothe peptides is a thiol and is conjugated to one or more of the peptidesof the composition through a disulfide bond.

In some embodiments, the linking moiety is hydrophilic such as, forexample, polyalkylene glycol. Before conjugation to the peptides of thecomposition, the hydrophilic linking moiety comprises at least tworeactive groups (X and Y), as described herein and as shown below:

In specific embodiments, the linking moiety is polyethylene glycol(PEG). The PEG in certain embodiments has a molecular weight of about200 Daltons to about 10,000 Daltons, e.g. about 500 Daltons to about5000 Daltons. The PEG in some embodiments has a molecular weight ofabout 10,000 Daltons to about 40,000 Daltons.

In some embodiments, the hydrophilic linking moiety comprises either amaleimido or an iodoacetyl group and an activated carboxylic acid (e.g.NHS ester) as the reactive groups. An example of a hydrophilic linkingmoiety comprising maleimido and NHS activating groups is shown below:

In some embodiments, the hydrophilic linking moiety comprises either amaleimido or an iodoacetyl group and a carboxylic acid as the reactivegroups. In these embodiments, the maleimido or iodoacetyl group can becoupled to a thiol moiety on a peptide (e.g. side chain of cysteine) andthe carboxylic acid can be coupled to a free amine on a peptide (e.g.side chain of lysine) with or without the use of a coupling reagent. Anyappropriate coupling agent known to one skilled in the art can be usedto couple the carboxylic acid with the free amine such as, for example,DCC, DIC, HATU, HBTU, and TBTU. For example, the hydrophilic linkingmoiety can comprise the following structure:

wherein n is 1 to 910.In exemplary embodiments, the linking moiety is maleimido-PEG(20kDa)-COOH, iodoacetyl-PEG(20 kDa)-COOH, maleimido-PEG(20 kDa)-NHS, oriodoacetyl-PEG(20 kDa)-NHS.

In some embodiments, the linking moiety is hydrophobic. Hydrophobiclinkers are known in the art. See, e.g., Bioconjugate Techniques, G. T.Hermanson (Academic Press, San Diego, Calif., 1996), which isincorporated by reference in its entirety. Suitable hydrophobic linkingmoieties are known in the art and include, for example,8-hydroxyoctanoic acid and 8-mercaptooctanoic acid. In specificembodiments, the hydrophilic linking moiety comprises an aliphatic chainof 2 to 100 methylene groups. Before conjugation to the peptides of thecomposition, the hydrophobic linking moiety comprises at least tworeactive groups (X and Y), as described herein and as shown below:

In some specific embodiments, the hydrophobic linking moiety compriseseither a maleimido or an iodoacetyl group and an activated carboxylicacid (e.g. NHS ester) as the reactive groups. An example of ahydrophobic linker comprising maleimido and NHS ester reactive groups isshown below:

In some specific embodiments, the hydrophobic linking moiety compriseseither a maleimido or an iodoacetyl group and a carboxylic acid. Inthese embodiments, the maleimido or iodoacetyl group can be coupled to athiol moiety on a peptide (e.g. side chain of cysteine) and thecarboxylic acid can be coupled to a free amine on a peptide (e.g. sidechain of lysine) with or without the use of a coupling reagent. Anycoupling agent known to one skilled in the art can be used to couple thecarboxylic acid with the free amine such as, for example, DCC, DIC,HATU, HBTU, and TBTU. For example, the hydrophobic linking moiety cancomprise the following structure:

wherein n is 1 to 500.

In some embodiments, the linking moiety is comprised of an amino acid, adipeptide, a tripeptide, or a polypeptide, wherein the amino acid,dipeptide, tripeptide, or polypeptide comprises at least two activatinggroups, as described herein.

Spacer

In some embodiments, the linking moiety is indirectly attached to one ormore peptides of the composition (e.g. the first peptide and/or secondpeptide) through a spacer. Before conjugation, the spacer isbifunctional and comprises two reactive groups. In some embodiments,both reactive groups of the spacer are the same or differentnucleophilic groups (e.g. amine, hydroxyl, thiol). In some embodiments,both reactive groups of the spacer are the same or differentelectrophilic groups (e.g. carboxyl group, activated form of a carboxylgroup, compound with a leaving group). In some embodiments, one reactivegroup of the spacer is nucleophilic and the other reactive group of thespacer is electrophilic. Nonlimiting combinations of the reactive groupson the spacer before conjugation are shown below.

Both Nucleophilic Both Electrophilic Nucleophilic/Electrophilic X Y X YX Y amino amino carboxyl carboxyl amino carboxyl amino thiol carboxylacyl chloride amino acyl chloride amino hydroxyl carboxyl anhydrideamino anhydride thiol amino carboxyl Ester amino ester thiol thiolcarboxyl NHS amino NHS thiol hydroxyl carboxyl Halogen amino halogenhydroxyl amino carboxyl sulfonate ester amino sulfonate ester hydroxylthiol carboxyl maleimido amino maleimido hydroxyl hydroxyl carboxylhaloacetyl amino haloacetyl carboxyl isocyanate amino isocyanate acylchloride carboxyl thiol carboxyl acyl chloride acyl chloride thiol acylchloride acyl chloride anhydride thiol anhydride acyl chloride Esterthiol ester acyl chloride NHS thiol NHS acyl chloride Halogen thiolhalogen acyl chloride sulfonate ester thiol sulfonate ester acylchloride maleimido thiol maleimido acyl chloride haloacetyl thiolhaloacetyl acyl chloride isocyanate thiol isocyanate anhydride carboxylhydroxyl carboxyl anhydride acyl chloride hydroxyl acyl chlorideanhydride anhydride hydroxyl anhydride anhydride Ester hydroxyl esteranhydride NHS hydroxyl NHS anhydride Halogen hydroxyl halogen anhydridesulfonate ester hydroxyl sulfonate ester anhydride maleimido hydroxylmaleimido anhydride haloacetyl hydroxyl haloacetyl anhydride isocyanatehydroxyl isocyanate ester carboxyl ester acyl chloride ester anhydrideester Ester ester NHS ester Halogen ester sulfonate ester estermaleimido ester haloacetyl ester isocyanate NHS carboxyl NHS acylchloride NHS anhydride NHS Ester NHS NHS NHS Halogen NHS sulfonate esterNHS maleimido NHS haloacetyl NHS isocyanate halogen carboxyl halogenacyl chloride halogen anhydride halogen Ester halogen NHS halogenHalogen halogen sulfonate ester halogen maleimido halogen haloacetylhalogen isocyanate sulfonate ester carboxyl sulfonate ester acylchloride sulfonate ester anhydride sulfonate ester Ester sulfonate esterNHS sulfonate ester Halogen sulfonate ester sulfonate ester sulfonateester maleimido sulfonate ester haloacetyl sulfonate ester isocyanatemaleimido carboxyl maleimido acyl chloride maleimido anhydride maleimidoEster maleimido NHS maleimido Halogen maleimido sulfonate estermaleimido maleimido maleimido haloacetyl maleimido isocyanate haloacetylcarboxyl haloacetyl acyl chloride haloacetyl anhydride haloacetyl Esterhaloacetyl NHS haloacetyl Halogen haloacetyl sulfonate ester haloacetylmaleimido haloacetyl haloacetyl haloacetyl isocyanate isocyanatecarboxyl isocyanate acyl chloride isocyanate anhydride isocyanate Esterisocyanate NHS isocyanate halogen isocyanate sulfonate ester isocyanatemaleimido isocyanate haloacetyl isocyanate isocyanateOther nonlimiting examples of reactive groups include pyridyldithiol,aryl azide, diazirine, carbodiimide, and hydrazide.

In some embodiments, the bifunctional spacer is hydrophilic. In certainembodiments, the reactive groups on the hydrophilic bifunctional spacerare a hydroxyl and a carboxylate. In other embodiments, the reactivegroups on the hydrophilic bifunctional spacer are an amine and acarboxylate. In other embodiments, the reactive groups on thehydrophilic bifunctional spacer are a thiol and a carboxylate. Inspecific embodiments, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments, the spacer is hydrophobic. Hydrophobic bifunctionalspacers are known in the art. See, e.g., Bioconjugate Techniques, G. T.Hermanson (Academic Press, San Diego, Calif., 1996), which isincorporated by reference in its entirety. In certain embodiments, thereactive groups on the hydrophobic bifunctional spacer are a hydroxyland a carboxylate. In other embodiments, the reactive groups on thehydrophobic bifunctional spacer are an amine and a carboxylate. In otherembodiments, the reactive groups on the hydrophobic bifunctional spacerare a thiol and a carboxylate. Suitable hydrophobic bifunctional spacerscomprising a carboxylate, and a hydroxyl group or a thiol group areknown in the art and include, for example, 8-hydroxyoctanoic acid and8-mercaptooctanoic acid.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol, aspreviously described herein (see Acylation and alkylation). The spacer(e.g., amino acid, dipeptide, tripeptide, hydrophilic bifunctional, orhydrophobic bifunctional spacer) in specific embodiments is 3 to 10atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or 10 atoms) in length.In more specific embodiments, the spacer is about 3 to 10 atoms (e.g., 6to 10 atoms) in length

In some aspects, one or more peptides of the conjugate (e.g. the firstpeptide and/or the second peptide) is linked to another peptide of thecomposition via the side chain of an internal amino acid. In someembodiments, the internal amino acid comprises a nucleophilic sidechain, such as an amino acid represented by Formula I, Formula II, orFormula III, as previously described herein (see Acylation andalkylation). In some embodiments, the internal amino acid is selectedfrom the group consisting of lysine, ornithine, serine, cysteine, andhomocysteine. In exemplary embodiments, the internal amino acid iscysteine. In some embodiments, the internal amino acid comprises anelectrophilic side chain such as, for example, Asp and Glu.

In some embodiments, the internal amino acid of one peptide of thecomposition (e.g. the first peptide) is linked to the C-terminal aminoacid of the other peptide of the composition (e.g. the second peptide).In some embodiments, the internal amino acid of one peptide of thecomposition (e.g. the first peptide) is linked to an internal amino acidof the other peptide of the composition (e.g. the second peptide).

In some embodiments, the internal amino acid of one peptide of thecomposition is directly linked (e.g. no linking moiety) to either aninternal amino acid or the C-terminal amino acid of another peptide ofthe composition. In some embodiments, the internal amino acid of onepeptide of the composition is linked to either an internal amino acid orthe C-terminal amino acid of another peptide of the composition througha linking moiety, as described herein. In some embodiments, the internalamino acid of one peptide of the composition is directly linked to thelinking moiety, as described herein. In some embodiments, the internalamino acid of one peptide of the composition is indirectly linked to thelinking moiety through a spacer, as described herein.

In some embodiments, the first and second peptide of the composition arelinked together to form a heterodimer. In some embodiments two analogsof the first peptide and or two analogs of the second peptide are linkedtogether to form a homodimer. In some exemplary embodiments, the sidechain of a cysteine residue at the C-terminus of the first peptide islinked to the side chain of a lysine residue at the C-terminus of thesecond peptide via a hydrophilic linker comprised of polyethyleneglycol. In some exemplary embodiments, the side chain of a cysteineresidue at position 24 of the first peptide is linked to the side chainof a lysine residue at the C-terminus of the second peptide via ahydrophilic linker comprised of polyethylene glycol, as shown below:

wherein n is 1 to 910.

In some exemplary embodiments, the side chain of a cysteine residue atposition 24 of the first peptide is linked to the side chain of a lysineresidue at the C-terminus of the second peptide via a hydrophobic linkercomprised of an aliphatic chain, as shown below:

wherein n is 1 to 500.

Compositions, Pharmaceutical Compositions

The present disclosures provide compositions comprising any of the GIPagonist peptides described herein and any of the glucagon antagonistpeptides described herein. In some embodiments, the compositioncomprises a GIP agonist peptide which exhibits at least 0.1% activity ofnative GIP at the GIP receptor and glucagon antagonist peptide whichexhibits at least 60% inhibition of the maximum response achieved byglucagon at the glucagon receptor. In some embodiments, the compositionis intended for therapeutic use in mammals, e.g., humans.

Pharmaceutical Salts

In some embodiments, the peptide(s) of the present disclosures (the GIPagonist peptide, the glucagon antagonist peptide, or both) is present inthe composition (or conjugate) in the form of a salt, e.g., apharmaceutically acceptable salt. As used herein the term“pharmaceutically acceptable salt” refers to salts of compounds thatretain the biological activity of the parent compound, and which are notbiologically or otherwise undesirable. Such salts can be prepared insitu during the final isolation and purification of the analog, orseparately prepared by reacting a free base function with a suitableacid. Many of the compounds disclosed herein are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Representative acid addition salts include,but are not limited to acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate,hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate, and undecanoate. Salts derived frominorganic acids include hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. Salts derived fromorganic acids include acetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid,maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluene-sulfonic acid, salicylic acid, and the like. Examples of acidswhich can be employed to form pharmaceutically acceptable acid additionsalts include, for example, an inorganic acid, e.g., hydrochloric acid,hydrobromic acid, sulphuric acid, and phosphoric acid, and an organicacid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.

Basic addition salts also can be prepared in situ during the finalisolation and purification of the source of salicylic acid, or byreacting a carboxylic acid-containing moiety with a suitable base suchas the hydroxide, carbonate, or bicarbonate of a pharmaceuticallyacceptable metal cation or with ammonia or an organic primary,secondary, or tertiary amine. Pharmaceutically acceptable salts include,but are not limited to, cations based on alkali metals or alkaline earthmetals such as lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like, and nontoxic quaternary ammonia and aminecations including ammonium, tetramethylammonium, tetraethylammonium,methylammonium, dimethylammonium, trimethylammonium, triethylammonium,diethylammonium, and ethylammonium, amongst others. Other representativeorganic amines useful for the formation of base addition salts include,for example, ethylenediamine, ethanolamine, diethanolamine, piperidine,piperazine, and the like. Salts derived from organic bases include, butare not limited to, salts of primary, secondary and tertiary amines.

Further, basic nitrogen-containing groups can be quaternized with thepeptide of the present disclosure (the GIP agonist peptide, the glucagonantagonist peptide, or both) as lower alkyl halides such as methyl,ethyl, propyl, and butyl chlorides, bromides, and iodides; long chainhalides such as decyl, lauryl, myristyl, and stearyl chlorides,bromides, and iodides; arylalkyl halides like benzyl and phenethylbromides and others. Water or oil-soluble or dispersible products arethereby obtained.

Formulations

In accordance with some embodiments, the composition of the presentdisclosures is a pharmaceutical composition and further comprises apharmaceutically acceptable carrier. The pharmaceutical composition cancomprise any pharmaceutically acceptable ingredient, including, forexample, acidifying agents, additives, adsorbents, aerosol propellants,air displacement agents, alkalizing agents, anticaking agents,anticoagulants, antimicrobial preservatives, antioxidants, antiseptics,bases, binders, buffering agents, chelating agents, coating agents,coloring agents, desiccants, detergents, diluents, disinfectants,disintegrants, dispersing agents, dissolution enhancing agents, dyes,emollients, emulsifying agents, emulsion stabilizers, fillers, filmforming agents, flavor enhancers, flavoring agents, flow enhancers,gelling agents, granulating agents, humectants, lubricants,mucoadhesives, ointment bases, ointments, oleaginous vehicles, organicbases, pastille bases, pigments, plasticizers, polishing agents,preservatives, sequestering agents, skin penetrants, solubilizingagents, solvents, stabilizing agents, suppository bases, surface activeagents, surfactants, suspending agents, sweetening agents, therapeuticagents, thickening agents, tonicity agents, toxicity agents,viscosity-increasing agents, water-absorbing agents, water-misciblecosolvents, water softeners, or wetting agents.

In some embodiments, the pharmaceutical composition comprises any one ora combination of the following components: acacia, acesulfame potassium,acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol,dehydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid,alginic acid, aliphatic polyesters, alumina, aluminum hydroxide,aluminum stearate, amylopectin, α-amylose, ascorbic acid, ascorbylpalmitate, aspartame, bacteriostatic water for injection, bentonite,bentonite magma, benzalkonium chloride, benzethonium chloride, benzoicacid, benzyl alcohol, benzyl benzoate, bronopol, butylatedhydroxyanisole, butylated hydroxytoluene, butylparaben, butylparabensodium, calcium alginate, calcium ascorbate, calcium carbonate, calciumcyclamate, dibasic anhydrous calcium phosphate, dibasic dehydratecalcium phosphate, tribasic calcium phosphate, calcium propionate,calcium silicate, calcium sorbate, calcium stearate, calcium sulfate,calcium sulfate hemihydrate, canola oil, carbomer, carbon dioxide,carboxymethyl cellulose calcium, carboxymethyl cellulose sodium,β-carotene, carrageenan, castor oil, hydrogenated castor oil, cationicemulsifying wax, cellulose acetate, cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, silicifiedmicrocrystalline cellulose, sodium carboxymethyl cellulose, cetostearylalcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol,chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidinegluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC),chlorodifluoromethane, chlorofluorocarbons (CFC)chlorophenoxyethanol,chloroxylenol, corn syrup solids, anhydrous citric acid, citric acidmonohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil,cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium,crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin,dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate,dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane(HFC), dimethyl-β-cyclodextrin, cyclodextrin-type compounds such asCaptisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate,disodium edentate, disodium hydrogen phosphate, docusate calcium,docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil, type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, potassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London,UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety, discloses various components used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

In some embodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration, such as, for example,at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v,1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60%w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoingcomponent(s) may be present in the pharmaceutical composition at anyconcentration, such as, for example, at most B, wherein B is 90% w/v,80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v,5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In otherembodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration range, such as, forexample from about A to about B. In some embodiments, A is 0.0001% and Bis 90%.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration. Incertain embodiments, the pharmaceutical compositions may comprisebuffering agents to achieve a physiological compatible pH. The bufferingagents may include any compounds capable of buffering at the desired pHsuch as, for example, phosphate buffers (e.g., PBS), triethanolamine,Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES,and others. In certain embodiments, the strength of the buffer is atleast 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, atleast 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least120 mM, at least 150 mM, or at least 200 mM. In some embodiments, thestrength of the buffer is no more than 300 mM (e.g., at most 200 mM, atmost 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM,at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10mM, at most 5 mM, at most 1 mM).

Routes of Administration

The following discussion on routes of administration is merely providedto illustrate exemplary embodiments and should not be construed aslimiting the scope in any way.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the peptide of the presentdisclosures (the GIP agonist peptide, the glucagon antagonist peptide,or both) dissolved in diluents, such as water, saline, or orange juice;(b) capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the peptide of the presentdisclosures (the GIP agonist peptide, the glucagon antagonist peptide,or both) in a flavor, usually sucrose and acacia or tragacanth, as wellas pastilles comprising the peptide of the present disclosure (the GIPagonist peptide, the glucagon antagonist peptide, or both) in an inertbase, such as gelatin and glycerin, or sucrose and acacia, emulsions,gels, and the like containing, in addition to, such excipients as areknown in the art.

The peptides of the disclosures (the GIP agonist peptide, the glucagonantagonist peptide, or both), alone or in combination with othersuitable components, can be delivered via pulmonary administration andcan be made into aerosol formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations also may be used to spray mucosa. In some embodiments, thepeptide (the GIP agonist peptide, the glucagon antagonist peptide, orboth) is formulated into a powder blend or into microparticles ornanoparticles. Suitable pulmonary formulations are known in the art.See, e.g., Qian et al., Int J Pharm 366: 218-220 (2009); Adjei andGarren, Pharmaceutical Research, 7(6): 565-569 (1990); Kawashima et al.,J Controlled Release 62(1-2): 279-287 (1999); Liu et al., Pharm Res10(2): 228-232 (1993); International Patent Application Publication Nos.WO 2007/133747 and WO 2007/141411.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous. The peptide of the present disclosure (the GIP agonistpeptide, the glucagon antagonist peptide, or both) can be administeredwith a physiologically acceptable diluent in a pharmaceutical carrier,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol or hexadecyl alcohol, a glycol, such as propylene glycol orpolyethylene glycol, dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the peptide of the present disclosure (the GIPagonist peptide, the glucagon antagonist peptide, or both) in solution.Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene glycol sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)).

Additionally, the peptide of the present disclosures (the GIP agonistpeptide, the glucagon antagonist peptide, or both) can be made intosuppositories for rectal administration by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the peptide of thedisclosures (the GIP agonist peptide, the glucagon antagonist peptide,or both) can be formulated as inclusion complexes, such as cyclodextrininclusion complexes, or liposomes.

Dose

The compositions of the present disclosures comprising a GIP agonistpeptide and a glucagon antagonist peptide, as described herein arebelieved to be useful in methods of treating a disease or medicalcondition in which glucagon receptor antagonism and GIP receptor agonism(and, optionally, GLP-1 receptor agonism) play a role. For purposes ofthe present disclosures, the amount or dose of the composition of thepresent disclosure administered should be sufficient to effect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the composition of thepresent disclosures should be sufficient to stimulate cAMP secretionfrom cells as described herein or sufficient to decrease blood glucoselevels, fat levels, food intake levels, or body weight of a mammal, in aperiod of from about 1 to 4 minutes, 1 to 4 hours or 1 to 4 weeks orlonger, e.g., 5 to 20 or more weeks, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular composition of thepresent disclosure and the condition of the animal (e.g., human), aswell as the body weight of the animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes herein, an assay, which comprises comparing the extent towhich blood glucose levels or body weight are lowered uponadministration of a given dose of the composition of the presentdisclosures to a mammal among a set of mammals of which is each given adifferent dose of the composition, could be used to determine a startingdose to be administered to a mammal. The extent to which blood glucoselevels or body weight are lowered upon administration of a certain dosecan be assayed by methods known in the art, including, for instance, themethods described herein as Examples 7-11.

Typically, the attending physician will decide the dosage of thecomposition of the present disclosure with which to treat eachindividual patient, taking into consideration a variety of factors, suchas age, body weight, general health, diet, sex, composition of thepresent disclosure to be administered, route of administration, severityof the condition being treated, and clinical effect to be achieved. Thedose of the composition of the present disclosure also will bedetermined by the existence, nature and extent of any adverse sideeffects that might accompany the administration of a particular peptideof the present disclosure.

Targeted Forms

One of ordinary skill in the art will readily appreciate that thepeptides of the compositions of the present disclosures can be modifiedin any number of ways, such that the therapeutic or prophylacticefficacy of the peptide (GIP agonist peptide, glucagon antagonistpeptide, or both) is increased through the modification. For instance,the peptide of the present disclosure (GIP agonist peptide, glucagonantagonist peptide, or both) can be conjugated either directly orindirectly through a linker to a targeting moiety. The practice ofconjugating compounds, e.g., peptides described herein (GIP agonistpeptide, glucagon antagonist peptide, or both), to targeting moieties isknown in the art. See, for instance, Wadhwa et al., J Drug Targeting, 3,111-127 (1995) and U.S. Pat. No. 5,087,616. The term “targeting moiety”as used herein, refers to any molecule or agent that specificallyrecognizes and binds to a cell-surface receptor, such that the targetingmoiety directs the delivery of the peptide of the present disclosures(GIP agonist peptide, glucagon antagonist peptide, or both) to apopulation of cells on which surface the receptor (the glucagonreceptor, the GIP receptor, the GLP-1 receptor) is expressed. Targetingmoieties include, but are not limited to, antibodies, or fragmentsthereof, peptides, hormones, growth factors, cytokines, and any othernatural or non-natural ligands, which bind to cell surface receptors(e.g., Epithelial Growth Factor Receptor (EGFR), T-cell receptor (TCR),B-cell receptor (BCR), CD28, Platelet-derived Growth Factor Receptor(PDGF), nicotinic acetylcholine receptor (nAChR), etc.). As used hereina “linker” is a bond, molecule or group of molecules that binds twoseparate entities to one another. Linkers may provide for optimalspacing of the two entities or may further supply a labile linkage thatallows the two entities to be separated from each other. Labile linkagesinclude photocleavable groups, acid-labile moieties, base-labilemoieties and enzyme-cleavable groups. The term “linker” in someembodiments refers to any agent or molecule that bridges the peptide ofthe present disclosures to the targeting moiety. One of ordinary skillin the art recognizes that sites on the peptide of the presentdisclosures (GIP agonist peptide, glucagon antagonist peptide, or both),which are not necessary for the function of the peptide of the presentdisclosures (GIP agonist peptide, glucagon antagonist peptide, or both),are ideal sites for attaching a linker and/or a targeting moiety,provided that the linker and/or targeting moiety, once attached to thepeptide of the present disclosures (GIP agonist peptide, glucagonantagonist peptide, or both), do(es) not interfere with the function ofthe peptide of the present disclosures (GIP agonist peptide, glucagonantagonist peptide, or both), i.e., the ability to stimulate cAMPsecretion from cells, to treat diabetes or obesity.

Controlled Release Formulations and Time of Administration

Alternatively, the peptides described herein (GIP agonist peptide,glucagon antagonist peptide, or both) can be modified into a depot form,such that the manner in which the peptide of the present disclosures isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of peptide of the present disclosures(GIP agonist peptide, glucagon antagonist peptide, or both) can be, forexample, an implantable composition comprising the peptide of thepresent disclosures and a porous or non-porous material, such as apolymer, wherein the peptide of the present disclosures is encapsulatedby or diffused throughout the material and/or degradation of thenon-porous material. The depot is then implanted into the desiredlocation within the body and the peptide of the present disclosures (GIPagonist peptide, glucagon antagonist peptide, or both) are released fromthe implant at a predetermined rate.

The pharmaceutical composition in certain aspects is modified to haveany type of in vivo release profile. In some aspects, the pharmaceuticalcomposition is an immediate release, controlled release, sustainedrelease, extended release, delayed release, or bi-phasic releaseformulation. Methods of formulating peptides for controlled release areknown in the art. See, for example, Qian et al., J Pharm 374: 46-52(2009) and International Patent Application Publication Nos. WO2008/130158, WO2004/033036; WO2000/032218; and WO 1999/040942.

The instant compositions may further comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect.

The disclosed pharmaceutical formulations may be administered accordingto any regime including, for example, daily (1 time per day, 2 times perday, 3 times per day, 4 times per day, 5 times per day, 6 times perday), every two days, every three days, every four days, every fivedays, every six days, weekly, bi-weekly, every three weeks, monthly, orbi-monthly.

Combinations

The peptides described herein (GIP agonist peptide, glucagon antagonistpeptide, or both) may be administered alone or in combination with othertherapeutic agents which aim to treat or prevent any of the diseases ormedical conditions described herein. For example, the peptides describedherein (GIP agonist peptide, glucagon antagonist peptide, or both) maybe co-administered with (simultaneously or sequentially) ananti-diabetic or anti-obesity agent. Anti-diabetic agents known in theart or under investigation include insulin, leptin, Peptide YY (PYY),Pancreatic Peptide (PP), fibroblast growth factor 21 (FGF21), Y2Y4receptor agonists, sulfonylureas, such as tolbutamide (Orinase),acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide(Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase,Glynase), glimepiride (Amaryl), or gliclazide (Diamicron); meglitinides,such as repaglinide (Prandin) or nateglinide (Starlix); biguanides suchas metformin (Glucophage) or phenformin; thiazolidinediones such asrosiglitazone (Avandia), pioglitazone (Actos), or troglitazone(Rezulin), or other PPARγ inhibitors; alpha glucosidase inhibitors thatinhibit carbohydrate digestion, such as miglitol (Glyset), acarbose(Precose/Glucobay); exenatide (Byetta) or pramlintide; Dipeptidylpeptidase-4 (DPP-4) inhibitors such as vildagliptin or sitagliptin; SGLT(sodium-dependent glucose transporter 1) inhibitors; glucokinaseactivators (GKA); glucagon receptor antagonists (GRA); or FBPase(fructose 1,6-bisphosphatase) inhibitors.

Anti-obesity agents known in the art or under investigation includeappetite suppressants, including phenethylamine type stimulants,phentermine (optionally with fenfluramine or dexfenfluramine),diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®),benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant(Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin;fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine),Excalia (bupropion and zonisamide) or Contrave (bupropion andnaltrexone); or lipase inhibitors, similar to XENICAL (Orlistat) orCetilistat (also known as ATL-962), or GT 389-255.

The peptides described herein (GIP agonist peptide, glucagon antagonistpeptide, or both) in some embodiments are co-administered with an agentfor treatment of non-alcoholic fatty liver disease or NASH. Agents usedto treat non-alcoholic fatty liver disease include ursodeoxycholic acid(a.k.a., Actigall, URSO, and Ursodiol), Metformin (Glucophage),rosiglitazone (Avandia), Clofibrate, Gemfibrozil, Polymixin B, andBetaine.

The peptides described herein (GIP agonist peptide, glucagon antagonistpeptide, or both) in some embodiments are co-administered with an agentfor treatment of a neurodegenerative disease, e.g., Parkinson's Disease.Anti-Parkinson's Disease agents are furthermore known in the art andinclude, but not limited to, levodopa, carbidopa, anticholinergics,bromocriptine, pramipexole, and ropinirole, amantadine, and rasagiline.

In view of the foregoing, the present disclosures further providepharmaceutical compositions and kits additionally comprising one ofthese other therapeutic agents. The additional therapeutic agent may beadministered simultaneously or sequentially with the peptide of thepresent disclosure. In some aspects, the peptide is administered beforethe additional therapeutic agent, while in other aspects, the peptide isadministered after the additional therapeutic agent.

Uses

Based on the information provided for the first time herein, it iscontemplated that the compositions (e.g., related pharmaceuticalcompositions) of the present disclosures are useful for treatment of adisease or medical condition, in which e.g., the lack of activity at theGIP receptor, the GLP-1 receptor, or at both receptors, is a factor inthe onset and/or progression of the disease or medical condition.Accordingly, the present disclosures provides a method of treating orpreventing a disease or medical condition in a patient, wherein thedisease or medical condition is a disease of medical condition in whicha lack of GIP receptor activation and/or GLP-1 receptor activation isassociated with the onset and/or progression of the disease of medicalcondition. The method comprises providing to the patient a compositionor conjugate in accordance with any of those described herein in anamount effective to treat or prevent the disease or medical condition.

In some embodiments, the disease or medical condition is metabolicsyndrome. Metabolic Syndrome, also known as metabolic syndrome X,insulin resistance syndrome or Reaven's syndrome, is a disorder thataffects over 50 million Americans. Metabolic Syndrome is typicallycharacterized by a clustering of at least three or more of the followingrisk factors: (1) abdominal obesity (excessive fat tissue in and aroundthe abdomen), (2) atherogenic dyslipidemia (blood fat disordersincluding high triglycerides, low HDL cholesterol and high LDLcholesterol that enhance the accumulation of plaque in the arterywalls), (3) elevated blood pressure, (4) insulin resistance or glucoseintolerance, (5) prothrombotic state (e.g., high fibrinogen orplasminogen activator inhibitor-1 in blood), and (6) pro-inflammatorystate (e.g., elevated C-reactive protein in blood). Other risk factorsmay include aging, hormonal imbalance and genetic predisposition.

Metabolic Syndrome is associated with an increased the risk of coronaryheart disease and other disorders related to the accumulation ofvascular plaque, such as stroke and peripheral vascular disease,referred to as atherosclerotic cardiovascular disease (ASCVD). Patientswith Metabolic Syndrome may progress from an insulin resistant state inits early stages to full blown type II diabetes with further increasingrisk of ASCVD. Without intending to be bound by any particular theory,the relationship between insulin resistance, Metabolic Syndrome andvascular disease may involve one or more concurrent pathogenicmechanisms including impaired insulin-stimulated vasodilation, insulinresistance-associated reduction in NO availability due to enhancedoxidative stress, and abnormalities in adipocyte-derived hormones suchas adiponectin (Lteif and Mather, Can. J. Cardiol. 20 (suppl. B):66B-76B(2004)).

According to the 2001 National Cholesterol Education Program AdultTreatment Panel (ATP III), any three of the following traits in the sameindividual meet the criteria for Metabolic Syndrome: (a) abdominalobesity (a waist circumference over 102 cm in men and over 88 cm inwomen); (b) serum triglycerides (150 mg/dl or above); (c) HDLcholesterol (40 mg/dl or lower in men and 50 mg/dl or lower in women);(d) blood pressure (130/85 or more); and (e) fasting blood glucose (110mg/dl or above). According to the World Health Organization (WHO), anindividual having high insulin levels (an elevated fasting blood glucoseor an elevated post meal glucose alone) with at least two of thefollowing criteria meets the criteria for Metabolic Syndrome: (a)abdominal obesity (waist to hip ratio of greater than 0.9, a body massindex of at least 30 kg/m2, or a waist measurement over 37 inches); (b)cholesterol panel showing a triglyceride level of at least 150 mg/dl oran HDL cholesterol lower than 35 mg/dl; (c) blood pressure of 140/90 ormore, or on treatment for high blood pressure). (Mathur, Ruchi,“Metabolic Syndrome,” ed. Shiel, Jr., William C., MedicineNet.com, May11, 2009).

For purposes herein, if an individual meets the criteria of either orboth of the criteria set forth by the 2001 National CholesterolEducation Program Adult Treatment Panel or the WHO, that individual isconsidered as afflicted with Metabolic Syndrome.

Without being bound to any particular theory, compositions andconjugates described herein are useful for treating Metabolic Syndrome.Accordingly, the invention provides a method of preventing or treatingMetabolic Syndrome, or reducing one, two, three or more risk factorsthereof, in a subject, comprising providing to the subject a compositiondescribed herein in an amount effective to prevent or treat MetabolicSyndrome, or the risk factor thereof.

In some embodiments, the method treats a hyperglycemic medicalcondition. In certain aspects, the hyperglycemic medical condition isdiabetes, diabetes mellitus type I, diabetes mellitus type II, orgestational diabetes, either insulin-dependent or non-insulin-dependent.In some aspects, the method treats the hyperglycemic medical conditionby reducing one or more complications of diabetes including nephropathy,retinopathy and vascular disease.

In some aspects, the disease or medical condition is obesity. In someaspects, the obesity is drug-induced obesity. In some aspects, themethod treats obesity by preventing or reducing weight gain orincreasing weight loss in the patient. In some aspects, the methodtreats obesity by reducing appetite, decreasing food intake, loweringthe levels of fat in the patient, or decreasing the rate of movement offood through the gastrointestinal system.

Because obesity is associated with the onset or progression of otherdiseases, the methods of treating obesity are further useful in methodsof reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases. The present disclosuresaccordingly provides methods of treating or preventing theseobesity-associated complications.

In some embodiments, the disease or medical condition is Nonalcoholicfatty liver disease (NAFLD). NAFLD refers to a wide spectrum of liverdisease ranging from simple fatty liver (steatosis), to nonalcoholicsteatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring ofthe liver). All of the stages of NAFLD have in common the accumulationof fat (fatty infiltration) in the liver cells (hepatocytes). Simplefatty liver is the abnormal accumulation of a certain type of fat,triglyceride, in the liver cells with no inflammation or scarring. InNASH, the fat accumulation is associated with varying degrees ofinflammation (hepatitis) and scarring (fibrosis) of the liver. Theinflammatory cells can destroy the liver cells (hepatocellularnecrosis). In the terms “steatohepatitis” and “steatonecrosis”, steatorefers to fatty infiltration, hepatitis refers to inflammation in theliver, and necrosis refers to destroyed liver cells. NASH can ultimatelylead to scarring of the liver (fibrosis) and then irreversible, advancedscarring (cirrhosis). Cirrhosis that is caused by NASH is the last andmost severe stage in the NAFLD spectrum. (Mendler, Michel, “Fatty Liver:Nonalcoholic Fatty Liver Disease (NAFLD) and NonalcoholicSteatohepatitis (NASH),” ed. Schoenfield, Leslie J., MedicineNet.com,Aug. 29, 2005).

Alcoholic Liver Disease, or Alcohol-Induced Liver Disease, encompassesthree pathologically distinct liver diseases related to or caused by theexcessive consumption of alcohol: fatty liver (steatosis), chronic oracute hepatitis, and cirrhosis. Alcoholic hepatitis can range from amild hepatitis, with abnormal laboratory tests being the only indicationof disease, to severe liver dysfunction with complications such asjaundice (yellow skin caused by bilirubin retention), hepaticencephalopathy (neurological dysfunction caused by liver failure),ascites (fluid accumulation in the abdomen), bleeding esophageal varices(varicose veins in the esophagus), abnormal blood clotting and coma.Histologically, alcoholic hepatitis has a characteristic appearance withballooning degeneration of hepatocytes, inflammation with neutrophilsand sometimes Mallory bodies (abnormal aggregations of cellularintermediate filament proteins). Cirrhosis is characterized anatomicallyby widespread nodules in the liver combined with fibrosis. (Worman,Howard J., “Alcoholic Liver Disease”, Columbia University Medical Centerwebsite).

Without being bound to any particular theory, the compositions andconjugates described herein are useful for the treatment of AlcoholicLiver Disease, NAFLD, or any stage thereof, including, for example,steatosis, steatohepatitis, hepatitis, hepatic inflammation, NASH,cirrhosis, or complications thereof. Accordingly, the presentdisclosures provides a method of preventing or treating Alcoholic LiverDisease, NAFLD, or any stage thereof, in a subject comprising providingto a subject a composition described herein in an amount effective toprevent or treat Alcoholic Liver Disease, NAFLD, or the stage thereof.Such treatment methods include reduction in one, two, three or more ofthe following: liver fat content, incidence or progression of cirrhosis,incidence of hepatocellular carcinoma, signs of inflammation, e.g.,abnormal hepatic enzyme levels (e.g., aspartate aminotransferase ASTand/or alanine aminotransferase ALT, or LDH), elevated serum ferritin,elevated serum bilirubin, and/or signs of fibrosis, e.g., elevatedTGF-beta levels. In certain embodiments, the compositions are used treatpatients who have progressed beyond simple fatty liver (steatosis) andexhibit signs of inflammation or hepatitis. Such methods may result, forexample, in reduction of AST and/or ALT levels.

GLP-1 and exendin-4 have been shown to have some neuroprotective effect.The present disclosures also provides uses of the compositions describedherein in treating neurodegenerative diseases, including but not limitedto Alzheimer's disease, Parkinson's disease, Multiple Sclerosis,Amylotrophic Lateral Sclerosis, other demyelination related disorders,senile dementia, subcortical dementia, arteriosclerotic dementia,AIDS-associated dementia, or other dementias, a central nervous systemcancer, traumatic brain injury, spinal cord injury, stroke or cerebralischemia, cerebral vasculitis, epilepsy, Huntington's disease,Tourette's syndrome, Guillain Barre syndrome, Wilson disease, Pick'sdisease, neuroinflammatory disorders, encephalitis, encephalomyelitis ormeningitis of viral, fungal or bacterial origin, or other centralnervous system infections, prion diseases, cerebellar ataxias,cerebellar degeneration, spinocerebellar degeneration syndromes,Friedreichs ataxia, ataxia telangiectasia, spinal dysmyotrophy,progressive supranuclear palsy, dystonia, muscle spasticity, tremor,retinitis pigmentosa, striatonigral degeneration, mitochondrialencephalo-myopathies, neuronal ceroid lipofuscinosis, hepaticencephalopathies, renal encephalopathies, metabolic encephalopathies,toxin-induced encephalopathies, and radiation-induced brain damage.

In some embodiments, the compositions are used in conjunction withparenteral administration of nutrients to non-diabetic patients in ahospital setting, e.g., to patients receiving parenteral nutrition ortotal parenteral nutrition. Nonlimiting examples include surgerypatients, patients in comas, patients with digestive tract illness, or anonfunctional gastrointestinal tract (e.g. due to surgical removal,blockage or impaired absorptive capacity, Crohn's disease, ulcerativecolitis, gastrointestinal tract obstruction, gastrointestinal tractfistula, acute pancreatitis, ischemic bowel, major gastrointestinalsurgery, certain congenital gastrointestinal tract anomalies, prolongeddiarrhea, or short bowel syndrome due to surgery, patients in shock, andpatients undergoing healing processes often receive parenteraladministration of carbohydrates along with various combinations oflipids, electrolytes, minerals, vitamins and amino acids. Thecompositions comprising the GIP agonist peptide and glucagon antagonistpeptide, as described herein, and the parenteral nutrition compositioncan be administered at the same time, at different times, before, orafter each other, provided that the composition is exerting the desiredbiological effect at the time that the parenteral nutrition compositionis being digested. For example, the parenteral nutrition may beadministered, 1, 2 or 3 times per day, while the composition isadministered once every other day, three times a week, two times a week,once a week, once every 2 weeks, once every 3 weeks, or once a month.

As used herein, the terms “treat,” and “prevent” as well as wordsstemming therefrom, do not necessarily imply 100% or complete treatmentor prevention. Rather, there are varying degrees of treatment orprevention of which one of ordinary skill hi the art recognizes ashaving a potential benefit or therapeutic effect. In this respect, themethods of the present disclosures can provide any amount of any levelof treatment or prevention of a disease or medical condition in amammal. Furthermore, the treatment or prevention provided by the methodcan include treatment or prevention of one or more conditions orsymptoms of the disease or medical condition. For example, with regardto methods of treating obesity, the method in some embodiments, achievesa decrease in food intake by or fat levels in a patient. Also, forpurposes herein, “prevention” can encompass delaying the onset of thedisease, or a symptom or condition thereof.

With regard to the above methods of treatment, the patient is any host.In some embodiments, the host is a mammal. As used herein, the term“mammal” refers to any vertebrate animal of the mammalia class,including, but not limited to, any of the monotreme, marsupial, andplacental taxas. In some embodiments, the mammal is one of the mammalsof the order Rodentia, such as mice and hamsters, and mammals of theorder Logomorpha, such as rabbits. In certain embodiments, the mammalsare from the order Carnivora, including Felines (cats) and Canines(dogs). In certain embodiments, the mammals are from the orderArtiodactyla, including Bovines (cows) and S wines (pigs) or of theorder Perssodactyla, including Equines (horses). In some instances, themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). In particular embodiments, themammal is a human.

Kits

The present disclosures further provide kits comprising a GIP agonistpeptide and glucagon antagonist peptide, wherein each of the GIP agonistpeptide and glucagon antagonist peptide are in accordance with theteachings found herein. Accordingly, in some embodiments, the kitcomprises a GIP agonist peptide which exhibits at least 0.1% activity ofnative GIP at the GIP receptor and a glucagon antagonist peptide whichexhibits at least 60% inhibition of the maximum response achieved byglucagon at the glucagon receptor.

In some aspects, the GIP agonist peptide is packaged separately from theglucagon antagonist peptide. For example, the kit may include twoseparate containers, e.g., vials, tubes, bottles, single ormulti-chambered pre-filled syringes, cartridges, infusion pumps(external or implantable), jet injectors, pre-filled pen devices and thelike, each of which contain one of the GIP agonist peptide and glucagonantagonist peptide. In alternative aspects, the GIP agonist peptide ispackaged together with the glucagon antagonist peptide, e.g., the GIPagonist peptide is conjugated to the glucagon antagonist peptide and theconjugate is provided in the kit in a single container, such as any ofthose described herein.

In some embodiments, the GIP agonist peptide, the glucagon antagonistpeptide, or both are provided in the kit as a lyophilized form or in anaqueous solution.

The kits in some embodiments comprise instructions for use. Theinstructions in some aspects include instructions for simultaneous andseparate co-administration of the GIP agonist peptide and glucagonantagonist peptide.

In one embodiment the kit is provided with a device for administeringthe composition to a patient, e.g., syringe needle, pen device, jetinjector or other needle-free injector. In accordance with oneembodiment the device of the kit is an aerosol dispensing device,wherein the composition is prepackaged within the aerosol device. Inanother embodiment the kit comprises a syringe and a needle, and in oneembodiment the sterile composition is prepackaged within the syringe.

In some embodiments, the kit comprises a pharmaceutically acceptablecarrier, such as any of those described herein.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1

Synthesis of Peptide Fragments of Glucagon

Materials:

All peptides described herein in the EXAMPLES were amidated unlessspecified otherwise.

MBHA resin (4-methylbenzhydrylamine polystyrene resin was used duringpeptide synthesis. MBHA resin, 100-180 mesh, 1% DVB cross-linkedpolystyrene; loading of 0.7-1.0 mmol/g), Boc-protected and Fmocprotected amino acids were purchased from Midwest Biotech. The solidphase peptide syntheses using Boc-protected amino acids were performedon an Applied Biosystem 430A Peptide Synthesizer. Fmoc protected aminoacid synthesis was performed using the Applied Biosystems Model 433Peptide Synthesizer.

Peptide Synthesis (Boc Amino Acids/HF Cleavage):

Synthesis of these analogs was performed on the Applied Biosystem Model430A Peptide Synthesizer. Synthetic peptides were constructed bysequential addition of amino acids to a cartridge containing 2 mmol ofBoc protected amino acid. Specifically, the synthesis was carried outusing Boc DEPBT-activated single couplings. At the end of the couplingstep, the peptidyl-resin was treated with TFA to remove the N-terminalBoc protecting group. It was washed repeatedly with DMF and thisrepetitive cycle was repeated for the desired number of coupling steps.After the assembly, the sidechain protection, Fmoc, was removed by 20%piperidine treatment and acylation was conducted using DIC. Thepeptidyl-resin at the end of the entire synthesis was dried by usingDCM, and the peptide was cleaved from the resin with anhydrous HF.

For the lactamization, orthogonal protecting groups were selected forGlu and Lys (e.g., Glu(Fm), Lys(Fmoc)). After removal of the protectinggroups and before HF cleavage, cyclization was performed as describedpreviously (see, e.g., International Patent Application Publication No.WO2008/101017).

HF Treatment of the Peptidyl-Resin

The peptidyl-resin was treated with anhydrous HF, and this typicallyyielded approximately 350 mg (˜50% yield) of a crudedeprotected-peptide. Specifically, the peptidyl-resin (30 mg to 200 mg)was placed in the hydrogen fluoride (HF) reaction vessel for cleavage.500 μL of p-cresol was added to the vessel as a carbonium ion scavenger.The vessel was attached to the HF system and submerged in themethanol/dry ice mixture. The vessel was evacuated with a vacuum pumpand 10 ml of HF was distilled to the reaction vessel. This reactionmixture of the peptidyl-resin and the HF was stirred for one hour at 0°C., after which a vacuum was established and the HF was quicklyevacuated (10-15 min). The vessel was removed carefully and filled withapproximately 35 ml of ether to precipitate the peptide and to extractthe p-cresol and small molecule organic protecting groups resulting fromHF treatment. This mixture was filtered utilizing a teflon filter andrepeated twice to remove all excess cresol. This filtrate was discarded.The precipitated peptide dissolves in approximately 20 ml of 10% aceticacid (aq). This filtrate, which contained the desired peptide, wascollected and lyophilized.

An analytical HPLC analysis of the crude solubilized peptide wasconducted under the following conditions [4.6×30 mm Xterra C8, 1.50mL/min, 220 nm, A buffer 0.1% TFA/10% ACN, B buffer 0.1% TFA/100% ACN,gradient 5-95% B over 15 minutes]. The extract was diluted twofold withwater and loaded onto a 2.2×25 cm Vydac C4 preparative reverse phasecolumn and eluted using an acetonitrile gradient on a Waters HPLC system(A buffer of 0.1% TFA/10% ACN, B buffer of 0.1% TFA/10% CAN and agradient of 0-100% B over 120 minutes at a flow of 15.00 ml/min. HPLCanalysis of the purified peptide demonstrated greater than 95% purityand electrospray ionization mass spectral analysis was used to confirmthe identity of the peptide.

Peptide Acylation

Acylated peptides were prepared as follows. Peptides were synthesized ona solid support resin using either a CS Bio 4886 Peptide Synthesizer orApplied Biosystems 430A Peptide Synthesizer. In situ neutralizationchemistry was used as described by Schnolzer et al., Int. J. PeptideProtein Res. 40: 180-193 (1992). For acylated peptides, the target aminoacid residue to be acylated (e.g., position ten, relative to the aminoacid position numbering of SEQ ID NO: 1) was substituted with an Nε-FMOC lysine residue. Treatment of the completed N-terminally BOCprotected peptide with 20% piperidine in DMF for 30 minutes removedFMOC/formyl groups. Coupling to the free ε-amino Lys residue wasachieved by coupling a ten-fold molar excess of either an FMOC-protectedspacer amino acid (ex. FMOC-Glu-OtBu) or acyl chain (ex.CH₃(CH₂)₁₄—COOH) and PyBOP or DEPBT coupling reagent in DMF/DIEA.Subsequent removal of the spacer amino acid's FMOC group is followed byrepetition of coupling with an acyl chain. Final treatment with 100% TFAresulted in removal of any side chain protecting groups and theN-terminal BOC group. Peptide resins were neutralized with 5% DIEA/DMF,dried, and then cleaved from the support using HF/p-cresol, 95:5, at 0°C. for one hour. Following ether extraction, a 5% HOAc solution was usedto solvate the crude peptide. A sample of the solution was then verifiedto contain the correct molecular weight peptide by ESI-MS. Correctpeptides were purified by RP-HPLC using a linear gradient of 10%CH3CN/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22 mm×250 mmprotein column was used for the purification. Acylated peptide analogsgenerally completed elution by a buffer ratio of 20:80. Portions werepooled together and checked for purity on an analytical RP-HPLC. Purefractions were lyophilized yielding white, solid peptides.

If a peptide comprised a lactam bridge and target residues to beacylated, acylation is carried out as described above upon addition ofthat amino acid to the peptide backbone.

Peptide PEGylation

For peptide PEGylation, 40 kDa methoxy poly(ethylene glycol)idoacetamide (NOF) was reacted with a molar equivalent of peptide in 7MUrea, 50 mM Tris-HCl buffer using the minimal amount of solvent neededto dissolve both peptide and PEG into a clear solution (generally lessthan 2 mL for a reaction using 2-3 mg peptide). Vigorous stirring atroom temperature commenced for 4-6 hours and the reaction analyzed byanalytical RP-HPLC. PEGylated products appeared distinctly from thestarting material with decreased retention times. Purification wasperformed on a Vydac C4 column with conditions similar to those used forthe initial peptide purification. Elution occurred around buffer ratiosof 50:50. Fractions of pure PEGylated peptide were found andlyophilized. Yields were above 50%, varying per reaction.

Analysis Using Mass Spectrometry

The mass spectra were obtained using a Sciex API-III electrosprayquadrapole mass spectrometer with a standard ESI ion source. Ionizationconditions that were used are as follows: ESI in the positive-ion mode;ion spray voltage, 3.9 kV; orifice potential, 60 V. The nebulizing andcurtain gas used was nitrogen flow rate of 0.9 L/min. Mass spectra wererecorded from 600-1800 Thompsons at 0.5 Th per step and 2 msec dwelltime. The sample (about 1 mg/mL) was dissolved in 50% aqueousacetonitrile with 1% acetic acid and introduced by an external syringepump at the rate of 5 μL/min.

When the peptides were analyzed in PBS solution by ESI MS, they werefirst desalted using a ZipTip solid phase extraction tip containing 0.6μL C4 resin, according to instructions provided by the manufacturer(Millipore Corporation, Billerica, Mass., see the Millipore website ofthe world wide web at millipore.com/catalogue.nsf/docs/C5737).

High Performance Liquid Chromatography (HPLC) Analysis:

Preliminary analyses were performed with these crude peptides to get anapproximation of their relative conversion rates in Phosphate BufferedSaline (PBS) buffer (pH, 7.2) using high performance liquidchromatography (HPLC) and MALDI analysis. The crude peptide samples weredissolved in the PBS buffer at a concentration of 1 mg/ml. 1 ml of theresulting solution was stored in a 1.5 ml HPLC vial which was thensealed and incubated at 37° C. Aliquots of 100 μl were drawn out atvarious time intervals, cooled to room temperature and analyzed by HPLC.

The HPLC analyses were performed using a Beckman System GoldChromatography system using a UV detector at 214 nm. HPLC analyses wereperformed on a 150 mm×4.6 mm C18 Vydac column. The flow rate was 1ml/min. Solvent A contained 0.1% TFA in distilled water, and solvent Bcontained 0.1% TFA in 90% CH3CN. A linear gradient was employed (40% to70% B in 15 minutes). The data were collected and analyzed using PeakSimple Chromatography software.

The initial rates of hydrolysis were used to measure the rate constantfor the dissociation of the respective prodrugs. The concentrations ofthe prodrug and the drug were estimated from their peak areasrespectively. The first order dissociation rate constants of theprodrugs were determined by plotting the logarithm of the concentrationof the prodrug at various time intervals. The slope of this plot givesthe rate constant ‘k’. The half lives of the degradation of the variousprodrugs were then calculated by using the formula t1/2=0.693/k.

In specific embodiments, the following procedures can be used:

General Peptide Synthesis Protocol with Boc-Chemistry Strategy:

Glucagon analogs were synthesized using HBTU-activated “Fast Boc” singlecoupling starting from 0.2 mmole of MBHA resin or first amino acidattached Pam resin on a modified Applied Biosystem 430A peptidesynthesizer. Boc amino acids and HBTU were obtained from Midwest Biotech(Fishers, Ind.). General side chain protecting groups used were:Arg(Tos), Asn(Xan), Asp(OcHex), Cys(pMeBzl), His(Bom), Lys(2Cl—Z),Ser(OBzl), Thr(OBzl), Tyr(2Br—Z), and Trp(CHO). Boc-Glu(OFm)-OH andBoc-Lys(Fmoc)-OH (Chem-Impex, Wood dale, IL) were used in thelactam-bridge formation sites. The N-terminal 3-phenyllactic acid (PLA)(Aldrich, Milwaukee, Wis.) was coupled manually by BEPBT(3-(Diethoxy-phosphoryloxy)-3H-benzo[d][1,2,3] triazin-4-one (SynchemInc., Aurora, Ohio) after the automated solid phase synthesis.

After peptide solid phase synthesis, each completed peptidyl resin wastreated with 20% piperidine/DMF to remove the Fmoc groups. For thelactam-bridge formation, usually 299 mg (1 mmole, 5-fold) BEPBT wereadded in 10% DIEA/DMF and reacted for 2˜4-h until ninhydrin test shownnegative.

Peptides were cleaved by liquid hydrogen fluoride cleavages performed inthe presence of p-cresol and dimethyl sulfide. The cleavage was run for1 hour in an ice bath using an HF apparatus (Penninsula Labs). Afterevaporation of the HF, the residue was suspended in diethyl ether andthe solid materials were filtered and washed with ether. Each peptidewas extracted into 30-70 ml aqueous acetic acid and diluted with waterand lyophilized. Crude peptide was analyzed by analytical HPLC andpeptide molecule weight was checked by ESI or MALDI-TOF massspectrometry. Peptides were then purified by the general HPLCpurification procedure.

General Peptide Synthesis Protocol with Fmoc-Chemistry Strategy:

Peptides were synthesized on an ABI 433A automated peptide synthesizerusing standard Fmoc chemistry with Rink MBHA amide resin or first aminoacid attached Wang resin (Novabiochem, San Diego, Calif.) using DIC/HOBTas coupling reagent. 3-phenyllactic acid (PLA) was coupled manually byBEPBT after the automated peptide synthesis. The side chain protectinggroups of N^(α)-Fmoc [N-(9-fluorenyl)methoxycarbonyl]amino acids were asfollows: Arg, Pmc; Asp, OtBu; Cys, Trt; Gln, Trt; His, Trt; Lys, Boc;Ser, tBu, Tyr, tBu; and Trp, Boc(Pmc=2,2,5,7,8-pentamethylchoman-6-sulfonyl, OtBu=tert-butyl ester,Trt=trityl, Boc=tert-butyloxycarbonyl, and tBu=tert-butyl ester).Fmoc-Glu(O-2-PhiPr)-OH and Fmoc-Lys(Mmt)-OH (Novabiochem, San Diego,Calif.) were incorporated in the lactam-bridge formation sites.

After solid phase synthesis, the 2-phenylisopropyl (2-PhiPr) group onthe Glu and the 4-methoxytrityl (Mmt) group on the Lys were removed byflashing 1% TFA/DCM though the peptidyl resin. For the lactam-bridgeformation, usually 150 mg (0.5 mmole, 5-fold) BEPBT were added in 10%DIEA/DMF and reacted for 2-4-h until ninhydrin test shown negative.

Peptides were cleaved from the resin with cleavage cocktail containing85% TFA, 5% phenol, 5% water and 5% thioanisole (2.5% EDT was added whenpeptide contains Cysteine). Crude peptides were precipitated in ether,centrifuged, and lyophilized. Peptides were then analyzed by analyticalHPLC and checked by ESI or MALDI-TOF mass spectrometry. Peptides werepurified by the general HPLC purification procedure.

General Analytical HPLC Procedure:

Analytical HPLC was performed on a Beckman System Gold HPLC system witha ZORBAX SB-C8 column (0.46×5 cm, 5 μm, Agilent) with a gradient elutionat a flow rate of 1.0 mL/min and monitored at 214 nm. The gradients wereset up as 10% B to 80% B over 10 min and then 10% B for 5 min. BufferA=0.1% TFA and B=0.1% TFA/90% acetonitrile.

General Preparative HPLC Purification Procedure:

If not specifically noted, the peptides were usually purified on aWaters 600E connected 486 monitor systems with semi-prepare HPLC column(ZORBAX SB-C8, 21.2×250 mm, 7 μm, Agilent) monitored at 214 nm or 230nM. Buffer A=0.1% TFA/10% acetonitrile and B=0.1% TFA/90% acetonitrile.The gradients used for the purification were 0-30% B over 40 min, then30-50% B over 30 min at a flow rate of 12 ml/min if not specificallynoted. Fractions were analyzed by analytical HPLC and checked by massspectrometry. The fractions over 90% pure were collected, lyophilizedand stored. The fractions with purity between 60-90% were combined,lyophilized and purified again.

General Pegylation Protocol: (Cys-maleimido)

Typically, the glucagon Cys analog is dissolved in phosphate bufferedsaline (5-10 mg/ml) and 0.01M ethylenediamine tetraacetic acid is added(10-15% of total volume). Excess (1.2˜2-fold) maleimidomethoxy-polyethylene glycol (MAL-m-dPEG) reagent is added and thereaction stirred at room temp while monitoring reaction progress byHPLC. After 2-12 h, the reaction mixture, is acidified and loaded onto apreparative reverse phase column for purification using 0.1%TFA/acetonitrile gradients. The appropriate fractions were combined andlyophilized to give the desired pegylated derivatives.

For peptides that exhibit low solubility in PBS, the peptides weredissolved in 25% acetonitrile water or 4˜6M urea buffer with 50˜100 mMTris (adjust pH 8.0˜8.5) and reacted with PEG reagents.

Specific examples of compounds synthesized by the methods describedabove are provided as follows:

Synthesis of [PLA6, D9, E16K20(lactam), D28]glucagon (6-29) amide

A peptide sequence TSDYSKYLDERRAKDFVQWLMDT (SEQ ID NO: 1249) was firstsolid phase synthesized on ABI 433A automated peptide synthesizer using0.1 mmole Fmoc/HOBT/DCC chemistry program with 0.1 mmole Rink MBHA amideresin using DIC/HOBT as coupling reagent. The following Fmoc amino acidwere used: Ala, Arg(Pmc), Asp(OtBu), Asn(Trt), Glu(O-2-PhiPr), Gln(Trt),Leu, Lys(Boc), Lys(Mmt), Met, PLA, Ser(tBu), Thr(tBu), Trp(Boc),Tyr(tBu), and Val. After the automated synthesis, the peptidyl resin wascoupled manually with 3-phenyllactic acid (83 mg, 0.5 mmole) and DEPBT(150 mg, 0.5 mmole) in 4 ml 5% DIEA/DMF for about 2 h to obtain thepeptidyl resin with the following sequence:HO-PLA-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-NH₂(SEQID NO: 1206).

Peptidyl resin was flashed with 50 ml 1% TFA/DCM in 5˜10 min and washedwith DCM, 5% DIEA/DMF and DMF. The peptidyl resin was then treated with150 mg (0.5 mmole, 5-fold) DEPBT in 10% DIEA/DMF for 2˜4 h untilninhydrin test shown negative.

Peptidyl resin was treated with 8.5 ml TFA with addition of 0.5 gphenol, 0.5 ml water and 0.5 ml thioanisole at room temperature forabout 2 h. The peptide dissolved in TFA was filtered and 40 ml ether wasadded to precipitate the peptide. The crude peptide were centrifuged,dissolved in aqueous acetic acid and lyophilized to get 150-250 mg crudepeptide. After purification 20˜30 mg (10˜15% yield totally) peptide with95% purity was obtained. The peptide was analyzed in general analyticalHPLC showing retention time as 7.63 min and ESI-MS analysis demonstratedthe desired mass of 2997.0 corresponding with the peptide molecularweight 2997.3.

Similar procedures were used to synthesize the following peptides:[PLA6, E9, E16K20(lactam)]glucagon (6-39) amide with analytical HPLC7.17 min and ESI-MS 3444.5 corresponding the calculated MW 3845.2;[PLA6, D9, K12E16(lactam), D28]glucagon (6-29) amide with analyticalHPLC 7.71 min and ESI-MS 2997.0 corresponding the calculated MW 2997.3;[PLA6, E9, K12E16(lactam)]glucagon (6-39) amide with analytical HPLC7.27 min and ESI-MS 3845.5 corresponding the calculated MW 3845.2;[PLA6, D9, E16K20(lactam), C24, D28]glucagon (6-29) amide withanalytical HPLC 7.85 min and ESI-MS 2972.0 corresponding the calculatedMW 2972.3; [PLA6, D9, K12E16(lactam), C24, D28]glucagon (6-29) amidewith analytical HPLC 7.83 min and ESI-MS 2971.5 corresponding thecalculated MW 2972.3; [PLA6, D9, E16K20(lactam), D28, C40]glucagon(6-40) amide with analytical HPLC 7.13 min and MALDI-MS 3935.7corresponding the calculated MW 3935.3.

Synthesis of [PLA6, D9, E16K20(lactam), C24(20K), D28]glucagon (6-29)amide

15 mg (0.005 mmole) [PLA6, D9, E16K20(lactam), C24, D28]glucagon (6-29)amide and 120 mg (0.006 mmole) 20K mPEG-MAL (MW ˜20k, ChirotechTechnology Ltd., Cambs CB4 0WG, German) were dissolved in 9 ml 25%acetonitrile water and about 0.5˜1 ml 1 M Tris base buffer (adjust pH to8.0˜8.5). The reaction was stirred at room temperature and the progressof the reaction was monitored by analytical HPLC. After no initialproduct was detected on HPLC (2˜6 h), the reaction mixture was directlypurified by preparative HPLC. The fractions were checked by analyticalHPLC at 214 nm and also measured by UV at 280 nm. The fractions with 90%HPLC purity and also with high absorption (A280 nm=1.0˜2.0) in UVmeasurement were combined and lyophilized. About 60˜80 mg [PLA6, D9,E16K20(lactam), C24(20K), D28]glucagon (6-29) amide can be obtainedwhich analytical HPLC analysis shown retention time as 8.5˜8.6 min andMALDI-MS shown broad mass spectrometry at 22K˜24K.

Similar procedures were used to synthesize [PLA6, D9, K12E16(lactam),C24(20K), D28]glucagon (6-29) amide and [PLA6, D9, E16K20(lactam), D28,C40(20K)]glucagon (6-40) amide.

Synthesis of Dimer[PLA6, D9, E16K20(lactam), C24, D28]glucagon (6-29)amide

20 mg (0.00673 mmole) [PLA6, D9, E16K20(lactam), C24, D28]glucagon(6-29) amide was dissolved in 6 ml PBS buffer, 0.5˜1 ml 1 M Tris base(adjust pH 8.0˜8.5) and 3 ml DMSO. The reaction mixture was stirred inan open air container and monitored by analytical HPLC every 2 h. Afterthe initial product (HPLC RT 7.85 min) was gone and the dimer product(HPLC RT 7.96 min) was the dominate product (˜24 h), the mixture wasdiluted with 0.1% TFA10% acetonitrile water and directly purified bypreparative HPLC. After lyophilized about 6-10 mg [PLA6, D9,E16K20(lactam), C24, D28]glucagon (6-29) amide was obtained with ESI-MS5942.0 corresponding the calculated MW 5942.6.

Synthesis of lactam-bridge depsipeptide [Aib2, E3, Thr5-O-PLA6,E16K20(lactam), D28]G(2-29) amide

A peptidyl resin with sequence HO-PLA-TSDYSKYLDERRAKDFVQWLMDT [PLA6,E16, K20, D28]glucagon (6-29) was synthesized by solid-phaseBoc-chemistry using an ABI 430A automated peptide synthesizer with 0.2mmole MBHA amide resin and DEPBT as coupling reagent. The following Bocamino acids were used: Ala, Arg(Tos), Asp(OcHx), Asn(Xan), Glu(OcHx),Gln(Xan), Leu, Lys(2-Cl—Z), Met, PLA, Ser(OBzl), Thr(OBzl), Trp(CHO),Tyr(2.6-di-Cl-Bzl) and Val except the glutamic acid at position 16 wasincorporated with Boc-Glu(OFm)-OH and lysine at position 20 wasincorporated with Boc-Lys(Fmoc)-OH. After removal of Fm and Fmocprotecting groups at position 16 and 20 with 20% piperidine in DMF, thepeptidyl resin was treated with 300 mg (1 mmol) DEPBT in 10% DIEA/DMFfor about 4 h to form the lactam bridge. To this lactam-bridged peptidylresin was added a pre-activated symmetrical anhydride solution composedof Boc-Thr(OBzl)-OH (2 mmol)/DIC (1 mmol)/DMAP (0.2 mmol) in DCM and thereaction was allowed to proceed for 16 h. The remaining amino acidsBoc-Gly-OH, Boc-Glu(OcHx)-OH and Boc-Aib-OH were coupled by standardBoc-chemistry again to obtain the depsipeptidyl resin of the followingsequence:Aib-Glu-Gly-Thr-O-PLA-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu*-Arg-Arg-Ala-Lys*-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-NH₂.(* are lactam bridged)

The peptidyl resin was treated with liquid hydrogen fluoride to cleavethe crude peptide from the solid support and remove all protectinggroups. The depsipeptide was purified by preparative HPLC, and analyzedby MS and analytical HPLC. The purified peptide demonstrated a singlepeak in analytical RP-HPLC and the ESI-MS analysis yielded the desiredmass of 3368.5 which corresponds with the calculated molecular weight of3369.0 daltons.

Similar procedures were used to synthesize the other lactam-bridgedepsipeptides reported in this patent.

Example 2

The ability of each peptide to induce cAMP was measured in a fireflyluciferase-based reporter assay. The cAMP production that is induced isdirectly proportional to the glucagon fragment binding to the glucagonreceptor or GIP receptor or GLP-1 receptor. HEK293 cells co-transfectedwith the receptor and luciferase gene linked to a cAMP responsiveelement were employed for the bioassay.

The cells were serum-deprived by culturing 16 hours in Dulbecco-modifiedMinimum Essential Medium (Invitrogen, Carlsbad, Calif.) supplementedwith 0.25% Bovine Growth Serum (HyClone, Logan, Utah) and then incubatedwith serial dilutions of glucagon fragments for 5 hours at 37° C., 5%CO2 in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences,San Jose, Calif.). At the end of the incubation, 100 μL of LucLiteluminescence substrate reagent (Perkin Elmer, Wellesley, Mass.) wereadded to each well. The plate was shaken briefly, incubated 10 min inthe dark and light output was measured on MicroBeta-1450 liquidscintillation counter (Perkin-Elmer, Wellesley, Mass.). The effective50% concentrations (EC50) and inhibitory 50% concentrations (IC50) werecalculated by using Origin software (OriginLab, Northampton, Mass.). AllEC50s and IC50s are reported in the following examples in nM, unlessindicated otherwise.

Example 3

The peptides listed in Table 1 were made as essentially described inExample 1 and tested for in vitro activity at each of the glucagonreceptor (for antagonist activity), GLP-1 receptor (for agonistactivity), and GIP receptor (for agonist activity) as essentiallydescribed in Example 2.

TABLE 1 SEQ ID Designa- Peptide Glucagon GLP-1 GIP NO: tion Monomer Name(IC50) (EC50) (EC50) 10 A1 PLA6, E9 G(6-29) ~20.0 — — 11 A2 PLA6, ~20.0~20.0 — (E16K20) G(6-29) 12 A3 HAibEGT-PLA6, 12.0 3.6 — (E16K20) G(1-29)13 A4 HAibEGT-PLA6, 14.3 0.19 — K10(rErEC16), (E16K20) G(1-29) 15 A5HAibEGT-PLA6, 150.0 0.76 — (E16K20), cex, K40(rErEC16) G(1-40) 17 B1 A1,Aib2, — — 3.89 E3 GIP(1-40) 18 B2 GIP-GLP(1-40)

Specifically, the heterodimers listed in Table 2 were made byconjugating Cys24 of the “A” peptide to Lys40 of the “B” peptide using abifunctional linker having a maleimido reactive group and anN-hydroxysuccinimide (NHS) ester reactive group flanking a methylenechain. After the “A” and “B” peptides were assembled and the side chainprotecting groups (Fmoc) were removed using 20% piperidine, thebifunctional linker was conjugated to Lys40 on the “B” peptide throughan acyl substitution reaction using a 4-fold excess of the linker in thepresence of diisopropylethylamine (DIEA, Step 1).

Step 1

The resulting peptide was treated with HF and purified using reversephase HPLC. This maleimido-functionalized peptide was dissolved in 50 mMTris buffer in the presence of urea (7 M, pH 8.5) and the “A” peptidewas introduced into the reaction mixture. The Cys24 of the “A” peptidereacted with the free maleimido group of the functionalized lysine in aMichael addition reaction to result in the heterodimer shown below (Step2).

Step 2

In some embodiments, these heterodimers can be synthesized using aheterobifunctional linker comprising a haloacetyl group instead of amaleimido group. In some embodiments, these heterodimers can besynthesized using a carboxylic acid with a coupling agent known to oneskilled in the art (e.g. DIC, TBTU, HATU, DCC, HBTU) instead of anactivated carboxylic acid such as the NHS ester.

The resulting heterodimers were tested for in vitro activity at each ofthe glucagon receptor (for inhibitory activity), GLP-1 activity (foragonist activity), and the GIP receptor (for agonist activity). The dataof these in vitro experiments are provided in Table 2.

TABLE 2 Glucagon GLP-1 GIP Dimer Name (IC50) (EC50) (EC50) NativeGlucagon (EC50) 0.126 Native GLP-1 0.04 Native GIP 0.011 A2-B1 PLA6,(E16K20), C24 G(6-29)/ 40.06 366.8 1.29 A1, Aib2, E3, K40 GIP(1-40)A4-B1 HAibEGT-PLA6, * 0.813 0.347 K10(rErEC16), (E16K20), C24G(1-29)/A1, Aib2, E3, K40 GIP(1-40) A1-B2 PLA6, E9, C24 G(6-29)/ 163.90.49 8.18 GIP-GLP(1-40)-K40 A3-B1 HAibEGT-PLA6, (E16K20), * 11.73 1.579C24 G(1-29)/A1, Aib2, E3, K40 GIP(1-40) A5-B1 HAibEGT-PLA6, (E16K20), *0.363 0.379 C24, cex, K40(rErEC16) G(1-40)/A1, Aib2, E3, K40 GIP(1-40)Values reported in nM; * Lost antagonist activity.

Example 4

The following peptides were made as essentially described in Example 1and tested in vitro for agonist activity at each of the GLP-1 receptor,glucagon receptor, and GIP receptor as essentially described in Example2:

Peptide mt-263 comprised a modified amino acid sequence of SEQ ID NO: 1in which position 1 was Tyr, position 2 was AIB, position 3 was Glu,position 12 was Ile, positions 16 to 18 were Lys, Gln, Ala,respectively, position 20 was AIB, position 21 was Glu, position 24 wasAsn, positions 27-29 were, Leu, Ala, and Gly, respectively, positions30-40 was GPSSGAPPPSK. The amino acid sequence of Peptide mt-263 isprovided herein as SEQ ID NO: 211.

Peptide mt-402 comprised the same amino acid sequence as that of mt-263,except, Gln at position 17 was changed to Lys and Glu at position 3 waschanged to Gln. The amino acid sequence of Peptide mt-402 is providedherein as SEQ ID NO: 25.

Peptide mt-403 comprised the same amino acid sequence as that of mt-402,except that the Lys at position 40 was covalently attached via itsepsilon amine to a C16 fatty acyl group. The amino acid sequence ofmt-403 is provided herein as SEQ ID NO: 26.

Peptide mt-404 comprised the same structure as mt-263, except that theGlu at position 3 was changed to a Gln, the Gln at position 17 waschanged to a Lys, and the AIB at position 20 was changed to a Glu. Theamino acid sequence of mt-404 is provided herein as SEQ ID NO: 27.

Peptide mt-405 comprised the same amino acid sequence as mt-404, exceptthat the Lys at position 40 was covalently attached via its epsilonamine to a C16 fatty acyl group. The amino acid sequence of mt-405 isprovided herein as SEQ ID NO: 28.

The EC50s at the GLP-1 receptor (GLP-1R), the glucagon receptor (GR),and the GIP receptor (GIPR) are provided in Table 3.

TABLE 3 GLP-1R GR GIPR relative relative relative Peptide EC₅₀, nMstandard activity EC₅₀, nM standard activity EC₅₀, nM standard activitymt-263 0.0100 0.0154 154.20% 4.0450 0.0762 1.88% 0.0054 0.0166 305.91%mt-402 0.0070 0.0154 220.60% 0.0298 0.0762 256.00% 0.0185 0.0166 89.46%mt-403 0.0027 0.0154 581.89% 0.0077 0.0762 987.18% 0.0061 0.0166 273.10%mt-404 0.0060 0.0154 258.29% 0.1076 0.0762 70.80% 0.0327 0.0166 50.57%mt-405 0.0022 0.0154 717.21% 0.0096 0.0762 797.18% 0.0039 0.0166 425.45%

The data suggest that acylation dramatically enhances activity at eachof the three receptors.

Example 5

Peptides mt-395 (SEQ ID NO: 33), mt-396 (SEQ ID NO: 34), mt-397 (SEQ IDNO: 35), and mt-398 (SEQ ID NO: 36) which were based on the structure ofmt-263 were made as essentially described in Example 1 and tested invitro as essentially described in Example 2. These peptides comprisedthe same structure as mt-263 except for the Lys at position 40 of mt-263was changed to Glu in mt-396, Arg in mt-397, d-Lys in mt-398, or deletedaltogether in mt-395. The EC50s at each of the GLP-1R, GR, and GIPR areshown in Table 4.

TABLE 4 GLP-1R GR GIPR relative relative relative Peptide EC₅₀, nMstandard activity EC₅₀, nM standard activity EC₅₀, nM standard activitymt-263 0.0081 0.0245 300.61% 3.1371 0.0298 0.95% 0.0033 0.0135 403.89%mt-395 0.0076 0.0245 321.55% 3.4095 0.0298 0.87% 0.0025 0.0135 537.45%mt-396 0.0093 0.0245 262.27% 2.9033 0.0298 1.03% 0.0034 0.0135 402.69%mt-397 0.0085 0.0245 287.88% 5.3528 0.0298 0.56% 0.0029 0.0135 470.03%mt-398 0.0078 0.0245 314.93% 3.7352 0.0298 0.80% 0.0031 0.0135 433.76%

These data suggest that Lys at position 40 can be a negative charged orpositive charged amino acid or can be deleted altogether and stillexhibit potent activity at the GLP-1R and GIPR.

Example 6

The following peptides were made as essentially described in Example 1and tested for agonist activity at each of the GLP-1R, GR, and GIPR asessentially described in Example 2.

Each of peptides mt-217 (SEQ ID NO: 19), mt-218 (SEQ ID NO: 20), mt-219(SEQ ID NO: 21), and mt-220 (SEQ ID NO: 22 comprised a modified aminoacid sequence of SEQ ID NO: 1 in which the following substitutions weremade: Tyr at position 1, AIB at position 2, Ile at position 12, Glu atposition 16, Gln at position 17, Ala at position 18, Lys at position 20,Glu at position 21, Lys at position 24, Phe at position 25, Leu-Ala-Glyat positions 27-29, and GPSSGAPPPS (SEQ ID NO: 3) at positions 30-39.The amino acids at positions 16 and 20 were bridged by a lactam. The Lysat position 24 of each of these peptides were attached to an amino acidspacer, which in turn, was attached to an acetylated Cys, which, inturn, was attached to a 40 kDa PEG. Peptide mt-217 had Ala as the aminoacid spacer, mt-218 had Glu as the amino acid spacer, mt-219 had Arg asthe amino acid spacer, and mt-220 had Phe as the amino acid spacer.

Peptides mt-225 (SEQ ID NO: 359), mt-226 (SEQ ID NO: 23), mt-227 (SEQ IDNO: 360), and mt-228 (SEQ ID NO: 24) comprised the same amino acidsequences as mt-217, mt-218, mt-219, and mt-220, respectively, exceptthat the modified Lys residue was at position 40 and the amino acid atposition 24 was changed to Asn.

The in vitro EC50s at each of the three receptors are shown in Table 5.

TABLE 5 GLP-1R GR GIPR relative relative relative Code EC₅₀, nM standardactivity EC₅₀, nM standard activity EC₅₀, nM Standard activity mt-2170.222 0.025 11.26% 13.886 0.114 0.82% 9.574 0.019 0.20% mt-218 0.3380.025 7.40% 16.298 0.114 0.70% 14.283 0.019 0.13% mt-219 0.151 0.02516.56% 17.628 0.114 0.65% 6.165 0.019 .31% mt-220 0.180 0.025 13.89%9.670 0.114 1.18% 10.268 0.019 0.19% mt-225 0.098 0.029 29.59% 2.7120.054 1.99% 1.899 0.017 0.90% mt-226 0.097 0.029 29.90% 3.462 0.0541.56% 1.467 0.017 1.16% mt-227 0.080 0.029 36.25% 4.244 0.054 1.27%1.320 0.017 1.29% mt-228 0.146 0.029 19.86% 5.364 0.054 1.01% 2.2660.017 0.75%

Example 7

Different doses of acylated peptides comprising an AIB at position 2,Glu at position 3, a Lys at position 16, and an AIB at position 20 weretested in vivo. The peptides were acylated at position 10 or position 40of the peptide, in the presence or absence of an acylation spacer. Morespecifically, peptides mt-261, mt-367, mt-270, and mt-369 at 1 or 10nmol/kg were subcutaneously injected daily for one week into mice(C57B1/6) aged 6 months old and having an initial body weight of 45 g.The mice had been on a diabetogenic diet for 4 months. Body weight, foodintake, blood glucose levels, and fat mass were measured during theexperiment. Each test group and control group consisted of 8 mice. Table6 provides the shorthand notation of each peptide indicating some of theamino acid modifications in the sequence, the SEQ ID NO: of the aminoacid sequence of each peptide and the % relative activity at each of theGLP-1R, GR, and GIPR as determined by the in vitro assay essentiallydescribed in Example 2.

TABLE 6 SEQ Position % Relative Activity ID Acyl of GLP- at at PeptideNO: Peptide Name Spacer Acyl 1R GR GIPR MT-261 205 E3K16AIB2, − 40 29932 298 20K40(C16) MT-367 335 E3K16AIB2, + 40 406 4 203 20K40(rErE- C16)MT-270 218 E3K16AIB2, − 10 212 0.1 163 20K10(C16) MT-369 337E3K16AIB2, + 10 385 2 204 20K10(rErE- C16) % relative activity is theactivity of the indicated peptide relative to the activity of the nativeligand at the indicated receptor.

As shown in FIG. 1, mice that were injected with 10 nmol/kg peptideexhibited a total change in body weight (%) (as calculated bysubtracting the body weight on Day 0 from the body weight on Day 7) ofat least −10%. A dramatic effect was observed with peptide MT-369. Thepresence of an acylation spacer had a bigger impact on the outcome(change in body weight) when the peptide was acylated at position 10, ascompared to when the peptide was acylated at position 40.

As shown in FIG. 2, mice that were injected with 10 nmol/kg peptidedemonstrated a total change in blood glucose levels (as calculated bysubtracting the blood glucose levels on Day 0 from that on Day 7) of atleast −40 mg/dL. The peptide which was acylated at position 10 with anacylation spacer decreased the blood glucose levels almost 100 mg/dL.

Example 8

Different doses of acylated peptides comprising a Lys at position 16 andan AIB at position 20 were tested in vivo. Specifically, peptidesMT-367, MT-369, MT-368, MT-384, MT-385, and MT-364 at 10 nmol/kg weresubcutaneously injected daily for one week into DIO mice (C57B1/6 WT)aged ˜10 months old and having an initial body weight of 57.6 g. Themice had been on a high fat diet for ˜8 months. Body weight and foodintake were measured on days 0, 1, 3, 5, and 7, whereas blood glucoselevels were measured on Days 0 and 7 of the experiment. Each test groupand control group consisted of 8 mice. Table 7 provides the shorthandnotation of each peptide indicating some of the amino acid modificationsin the sequence, the SEQ ID NO: of the amino acid sequence of eachpeptide and the % relative activity at each of the GLP-1R, GR, and GIPRas determined by the in vitro assay essentially described in Example 2.

TABLE 7 SEQ ID Acyl Position of % Relative Activity Peptide NO: PeptideName Spacer Acyl GLP-1R at GR at GIPR MT-367 335 E3K16AIB20K40(rErE- +40 406 4 203 C16) MT-369 336 E3K16AIB20K10(rErE- + 10 385 2 204 C16)MT-368 337 Q3K16AIB20K40(rErE- + 40 419 492 296 C16) MT-384 29Q3K16AIB20K10(rErE- + 10 349 228 808 C16) MT-385 31Q3I7K16AIB20K10(rErE- + 10 3 239 715 C16) MT-364 1069Chimera2-CEXK40(C16) − 40 355 350 16 % Relative Activity is activity ofthe peptide at the indicated receptor relative to the native ligand ofthat receptor.

As shown in FIG. 3, mice injected with a peptide which exhibited invitro activity at the GLP-1 receptor demonstrated a total change in bodyweight (%) of at least 18%, whereas the group of mice that were injectedwith MT-385 (comprising an Ile at position 7, which decreases GLP-1 Ractivity) exhibited a lower change in body weight, thereby demonstratingthe importance of the GLP-1 agonist aspect of the peptide.

As shown in FIG. 4, mice injected with MT-385, MT-364, MT-384 or MT-369exhibited a total change in blood glucose levels of greater than −50mg/dL.

Example 9

Peptides comprising a Glu or Gln at position 3, Lys at position 16, Glnat position 17, Ala at position 18, and an AIB at positions 2 and 20,along with a Glu at position 21, Asn at position 24, and a C-terminalextension of GPSSGAPPPSK were made and tested in vivo as essentiallydescribed in the previous Examples.

More specifically, peptides having the amino acid sequences of the SEQID NOs: indicated in Table 8 were made. None of the peptides comprisedan acyl group. C57B1/6 mice with an initial body weight of 55 g weresubcutaneously injected daily with 30 nmol/kg for one week. The micewere 10 months old and had been on a diabetogenic diet for 8 months.Body weight, food intake, blood glucose levels and fat mass weremonitored during the course of the study.

Table 8 provides the shorthand notation of each peptide indicating someof the amino acid modifications in the sequence, the SEQ ID NO: of theamino acid sequence of each peptide and the % relative activity at eachof the GLP-1R, GR, and GIPR as determined by the in vitro assayessentially described in Example 2.

TABLE 8 SEQ ID % Relative Activity Peptide NO: Peptide Name GLP-1R at GRat GIPR Exendin-4- 37 312 0.01 0.01 like peptide MT-263 211Y1Aib2E3I12K16Q17A18Aib20E21N24- 169 0.74 225 LAG27-29-CEX-K MT-280 226Y1Aib2I12K16Q17A18Aib20E21N24- 225 60 154 LAG27-29-CEX-K MT-356 332Y1Aib2E3I12K16Q17A18Aib20E21N24- 221 82 25 CEX-K MT-357 333Y1Aib2I7I12K16Q17A18Aib20E21N24- 0.81 170 198 LAG27-29-CEX-K

As shown in FIG. 5, mice injected with MT-263 comprising a Glu atposition 3 demonstrated a significant weight loss over the course of the7-day study. This peptide achieved levels of weight loss substantiallygreater than the Exendin-4-like positive control peptide.

As shown in FIG. 6, MT-263 achieved the greatest change in blood glucoselevels over the course of the 7-day study. Mice injected with MT-280also demonstrated a significant decrease in blood glucose levels.

Example 10

The pharmokinetic properties of Peptide MT-263 were tested by varyingthe administration regimen. Diet-induced obesity (DIO) mice (N=8, 10mice per group) were injected during the course of the 6 day study asfollows:

Groups A and B: daily injections of 10 nmol/kg MT-263 in vehicle;

Group C: injections every other day with 20 nmol/kg MT-263 in vehicle;

Groups D and E: injections every 3 days with 30 nmol/kg of MT-263 invehicle.

As shown in FIG. 7, the % change in body weight of mice injected withpeptide on a daily basis exhibited a steady weight loss over the courseof the six day study.

As shown in FIG. 8, the biggest decrease in blood glucose levels wasobserved in mice injected with MT-263 on a daily basis.

Example 11

Acylated or pegylated compounds were subcutaneously injected into DIOmice (N=8; 7 mice per group) having an initial body weight of 57.4 g.The acylated compounds were injected daily at a dose of 10 nmol/kg,whereas the pegylated compounds were injected QW at a dose of 10nmol/kg. Body weight, food intake, blood glucose levels, and fat masswere monitored throughout the study.

Table 9 provides the in vitro activities and structural features of thepeptides used in this study.

TABLE 9 SEQ % Relative Activity Peptide ID NO: Peptide Name GLP-1R at GRat GIPR MT-270 218 E3K10-C16 211.8 0.06 163.3 MT-341 262E3C24-PEG-K10-C14 14.6 0.01 27.4 MT-261 205 E3K40-C16 372.3 13.4 700MT-353 266 E3C24-PEG-K40-C14 128.6 0.17 102.9 MT-278 224 K40-C16 459.5588.4 846.4 MT-290 236 C24-PEG-K40-C14 156.4 113.8 23.7

FIGS. 40 and 41 demonstrate the results of the changes in body weightand blood glucose levels upon administration of the peptides.

Example 12

Peptides MT-261 (SEQ ID NO: 205) and MT-278 (SEQ ID NO: 224) (at doses:0.3, 1, 3, or 10 nmol/kg/day) were administered to mice as essentiallydescribed herein and body weight, food intake, and blood glucose levelswere monitored. FIGS. 42 and 43 demonstrate the results of the changesin body weight and blood glucose levels, respectively.

Example 13

Several peptides were made as essentially described herein and thestructures of each can be found in Sequence Listing 2. The in vivoeffects of each peptide were tested in mice as essentially describedherein. FIGS. 1-31 provide the results of the in vivo assays.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A peptide combination comprising a GIP agonist peptide which exhibitsat least 0.1% activity of native GIP at the GIP receptor and a glucagonantagonist peptide which exhibits at least 60% inhibition of the maximumresponse achieved by glucagon at the glucagon receptor.
 2. The peptidecombination of claim 1, wherein the GIP agonist peptide and the glucagonantagonist peptide are components of a composition.
 3. The peptidecombination of claim 2, wherein the composition is a pharmaceuticalcomposition further comprising a pharmaceutically acceptable carrier. 4.The peptide combination of claim 1, wherein the GIP agonist peptide andthe glucagon antagonist peptide are components of a kit.
 5. The peptidecomposition of claim 4, wherein the kit further comprises instructionsof using the GIP peptide and glucagon antagonist peptide for treating amammal.
 6. The peptide combination of claim 4 or 5, wherein the GIPagonist peptide is packaged separately from the glucagon antagonistpeptide in the kit.
 7. The peptide combination of claim 4 or 5, whereinthe GIP agonist peptide is packaged with the glucagon antagonist peptidein the kit.
 8. The peptide combination of claim 1, wherein the GIPagonist peptide is attached to the glucagon antagonist peptide to form aconjugate.
 9. The peptide combination of claim 8, wherein the GIPagonist peptide is covalently attached to the glucagon antagonistpeptide.
 10. The peptide combination of claim 9, wherein the conjugateis a fusion peptide comprising the GIP agonist peptide and the glucagonantagonist peptide.
 11. The peptide combination of claim 9, wherein theconjugate is a heterodimer comprising the GIP agonist peptide linked tothe glucagon antagonist peptide.
 12. The peptide combination of claim11, wherein the heterodimer comprises a linker which connects the GIPagonist peptide linked to the glucagon antagonist peptide.
 13. Thepeptide combination of claim 12, wherein the linker is a bifunctionallinker.
 14. The peptide combination of claim 13, wherein thebifunctional linker comprises a polyethylene glycol.
 15. The peptidecombination of claim 13 or 14, wherein one end of the bifunctionallinker is attached to a Lys of one of the GIP agonist peptide andglucagon antagonist peptide and the other end of the bifunctional linkeris attached to a Cys of the other peptide.
 16. The peptide combinationof claim 15, wherein the Lys is located at the C-terminus of the GIPagonist peptide.
 17. The peptide combination of claim 15 or 16, whereinthe Cys is located at the C-terminus or at position 24 of the glucagonantagonist peptide.
 18. The peptide combination of any of the precedingclaims, wherein the GIP agonist peptide exhibits at least 10% of theactivity of native GIP at the GIP receptor.
 19. The peptide combinationof any of the preceding claims, wherein the GIP agonist peptide exhibitsno more than 1% of the activity of native glucagon at the glucagonreceptor.
 20. The peptide combination of any of the preceding claims,wherein the glucagon antagonist peptide exhibits at least 80% inhibitionof the maximum response achieved by glucagon at the glucagon receptor.21. The peptide combination of any of the preceding claims, wherein theEC50 at the GIP receptor of the GIP agonist peptide is within about500-fold of the IC50 at the glucagon receptor of the glucagon antagonistpeptide.
 22. The peptide combination of any of the preceding claims,wherein (i) the GIP agonist peptide activates the GLP-1 receptor; (ii)wherein the glucagon antagonist peptide activates the GLP-1 receptor; or(iii) both (i) and (ii).
 23. The peptide combination of any of thepreceding claims, wherein the GIP agonist peptide is an analog of nativehuman GIP (SEQ ID NO: 2) with up to 15 amino acid modifications.
 24. Thepeptide combination of claim 23, wherein the GIP agonist peptidecomprises amino acids 3-28 of native human GIP (SEQ ID NO: 2).
 25. Thepeptide combination of claim 24, wherein the GIP agonist peptidecomprises alpha-aminoisobutyric acid (AIB) N-terminal to amino acids3-28 of native human GIP.
 26. The peptide combination of claim 25,wherein the GIP agonist peptide comprises a small aliphatic amino acidN-terminal to the AIB.
 27. The peptide combination of any of claims1-22, wherein (i) the GIP agonist peptide is an analog of native humanglucagon (SEQ ID NO: 1) with up to 15 amino acid modifications; (ii) theglucagon antagonist peptide is an analog of native human glucagon (SEQID NO: 1) with up to 15 amino acid modifications; or (iii) both (i) and(ii).
 28. The peptide combination of claim 27, wherein the GIP agonistpeptide is an analog of native human glucagon (SEQ ID NO: 1) comprising:(a) an amino acid modification at position 1 that confers GIP agonistactivity, (b) a modification selected from the group consisting of: (i)a lactam bridge between the side chains of amino acids at positions iand i+4 or between the side chains of amino acids at positions j andj+3, wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17, and(ii) one, two, three, or all of the amino acids at positions 16, 20, 21,and 24 of the analog is substituted with an α,α-disubstituted aminoacid, (c) 1-10 further amino acid modifications, and (d) optionally, aC-terminal amide.
 29. The peptide combination of claim 28, wherein theamino acid modification at position 1 is a substitution of His with anamino acid lacking an imidazole side chain.
 30. The peptide combinationof claim 29, wherein the amino acid lacking an imidazole side chain is alarge, aromatic amino acid.
 31. The peptide combination of claim 30,wherein the large, aromatic amino acid is Tyr.
 32. The peptidecombination of claim 30, wherein the amino acid lacking an imidazoleside chain is a small aliphatic amino acid.
 33. The peptide combinationof claim 32, wherein the small aliphatic amino acid is Ala.
 34. Thepeptide combination of any of claims 28 to 33, wherein the lactam bridgeis between the amino acids at positions 16 and 20, wherein one of theamino acids at positions 16 and 20 is substituted with Glu, and theother of the amino acids at positions 16 and 20 is substituted with Lys.35. The peptide combination of any of claims 28 to 34, wherein theα,α-disubstituted amino acid is AIB.
 36. The peptide combination of anyof claims 28 to 35, wherein the amino acid at position 16 or position 20is substituted with an α,α-disubstituted amino acid.
 37. The peptidecombination of claim 36, wherein the amino acid at position 20 is AIBand the amino acid at position 16 is substituted with a positive-chargedamino acid.
 38. The peptide combination of claim 37, wherein thepositive-charged amino acid is an amino acid of Formula IV:

wherein n is 1 to 7, wherein each of R1 and R2 is independently selectedfrom the group consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group.
 39. Thepeptide combination of claim 38, wherein the amino acid of Formula IV ishomoLys, Lys, Orn, or 2,4-diaminobutyric acid (Dab).
 40. The peptidecombination of any of claims 27 to 39, wherein the GIP agonist peptidecomprises amino acid modifications at one, two or all of positions 27,28 and
 29. 41. The peptide combination of claim 40, wherein (a) the Metat position 27 of the GIP agonist peptide is substituted with a largealiphatic amino acid, optionally Leu, (b) the Asn at position 28 of theGIP agonist peptide is substituted with a small aliphatic amino acid,optionally Ala, (c) the Thr at position 29 of the GIP agonist peptide issubstituted with a small aliphatic amino acid, optionally Gly, or (d) acombination of two or all of (a), (b), and (c).
 42. The peptidecombination of claim 41, wherein the GIP agonist peptide comprises Leuat position 27, Ala at position 28, and Gly or Thr at position
 29. 43.The peptide combination of any of claims 27 to 42 wherein the GIPagonist peptide comprises an extension of 1 to 21 amino acids C-terminalto the amino acid at position 28 or
 29. 44. The peptide combination ofclaim 43, wherein the extension comprises the amino acid sequence of SEQID NO: 3 or
 4. 45. The peptide combination of claim 43 or 44, wherein1-6 amino acids of the extension are positive-charged amino acids. 46.The peptide combination of claim 45, wherein the 1-6 positive-chargedamino acids comprises Arg or amino acids of Formula IV:

wherein n is 1 to 7, wherein each of R1 and R2 is independently selectedfrom the group consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group.
 47. Thepeptide combination of claim 46, wherein the amino acid of Formula IV ishomoLys, d-Lys, Lys, Orn, or Dab.
 48. The peptide combination of any ofclaims 27 to 47, wherein the GIP agonist peptide comprises an amino acidcomprising a side chain covalently linked to an acyl or alkyl group,which acyl or alkyl group is non-native to a naturally-occurring aminoacid.
 49. The peptide combination of claim 48, wherein the amino acidlinked to the acyl or alkyl group is an amino acid of Formula I, FormulaII, or Formula III.
 50. The peptide combination of claim 49, wherein theamino acid of Formula I is Lys.
 51. The peptide combination of any ofclaims 48 to 50, wherein the amino acid linked to the acyl or alkylgroup is located at position 10 of the of the GIP agonist peptiderelative to SEQ ID NO:
 1. 52. The peptide combination of any of claims48 to 51, wherein, when the GIP agonist peptide comprises an extensionof 1 to 21 amino acids C-terminal to the amino acid at position 28 or29, the amino acid linked to the acyl or alkyl group is located at aposition corresponding to any of positions 37-43.
 53. The peptidecombination of claim 52, wherein the amino acid linked to the acyl oralkyl group is located at position
 40. 54. The peptide combination ofany of claims 48 to 53, wherein the acyl or alkyl group is covalentlyattached to the side chain of the amino acid via a spacer.
 55. Thepeptide combination of claim 54, wherein the spacer is 3 to 10 atoms inlength.
 56. The peptide combination of claim 55, wherein the spacer isan amino acid or dipeptide.
 57. The peptide combination of claim 56,wherein the spacer is 6-amino hexanoic acid.
 58. The peptide combinationof claim 56, wherein the spacer is a dipeptide comprising two acidicamino acids.
 59. The peptide combination of any of claims 54 to 58,wherein the total length of the spacer and the acyl group is about 14 toabout 28 atoms in length.
 60. The peptide combination of any of claims48 to 59, wherein the acyl group is a C12 to C18 fatty acyl group. 61.The peptide combination of claim 60, wherein the acyl group is a C14 orC16 fatty acyl group.
 62. The peptide combination of any of claims 27 to61, wherein the GIP agonist peptide comprises a modification selectedfrom the group consisting of: (a) Ser at position 2 substituted withD-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val, or α-amino-N-butyricacid; (a) Tyr at position 10 substituted with Trp, Lys, Orn, Glu, Phe,or Val; (b) Linkage of an acyl group to a Lys at position 10; (c) Lys atposition 12 substituted with Arg or Ile; (d) Ser at position 16substituted with Glu, Gln, homoglutamic acid, homocysteic acid, Thr,Gly, or AIB; (e) Arg at position 17 substituted with Gln; (f) Arg atposition 18 substituted with Ala, Ser, Thr, or Gly; (g) Gln at position20 substituted with Ser, Thr, Ala, Lys, Citrulline, Arg, Orn, or AIB;(h) Asp at position 21 substituted with Glu, homoglutamic acid,homocysteic acid; (i) Val at position 23 substituted with Ile; (j) Glnat position 24 substituted with Asn, Ser, Thr, Ala, or AIB; and (k) aconservative substitution at any of positions 2, 5, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28, and
 29. 63. The peptidecombination of any of claims 1-22, wherein the GIP agonist peptide is ananalog of glucagon (SEQ ID NO: 1) comprising an acyl group, wherein theacyl group is attached to a spacer, wherein: (i) the spacer is attachedto the side chain of the amino acid at position 10 of the analog; or(ii) the analog comprises an extension of 1 to 21 amino acids C-terminalto the amino acid at position 29 and the spacer is attached to the sidechain of an amino acid corresponding to one of positions 37-43 relativeto SEQ ID NO: 1; wherein the analog exhibits at least 1% activity ofnative GIP at the GIP receptor.
 64. The peptide combination of claim 63,comprising an amino acid sequence of SEQ ID NO: 1 with (i) an amino acidmodification at position 1 that confers GIP agonist activity and (ii) atleast one or both of: (A) the analog comprises a lactam bridge betweenthe side chains of amino acids at positions i and i+4 or between theside chains of amino acids at positions j and j+3, wherein i is 12, 13,16, 17, 20 or 24, and wherein j is 17; and (B) one, two, three, or allof the amino acids at positions 16, 20, 21, and 24 of the analog issubstituted with an α,α-disubstituted amino acid; and (iii) up to 6further amino acid modifications
 65. The peptide combination of claim64, wherein the analog comprises (i) an amino acid substitution of Serat position 16 with an amino acid of Formula IV:

wherein n is 1 to 7, wherein each of R1 and R2 is independently selectedfrom the group consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group; and (ii) anamino acid substitution of the Gln at position 20 with an alpha,alpha-disubstituted amino acid.
 66. The peptide combination of any ofclaims 63 to 65, comprising amino acid modifications at one, two, or allof positions 27, 28, and
 29. 67. The peptide combination of claim 66,comprising Leu at position 27, Ala at position 28, and Gly or Thr atposition
 29. 68. The peptide combination of any of claims 63 to 67,wherein the extension comprises the amino acid sequence of SEQ ID NO: 3or
 4. 69. The peptide combination of any of claims 63 to 68, wherein thespacer is attached to the side chain of an amino acid of Formula I,Formula II, or Formula III.
 70. The peptide combination of claim 69,wherein the amino acid of Formula I is Lys.
 71. The peptide combinationof any of claims 63 to 70, wherein the spacer is 3 to 10 atoms inlength.
 72. The peptide combination of claim 71, wherein the spacer isan amino acid or dipeptide.
 73. The peptide combination of claim 72,wherein the spacer is 6-amino hexanoic acid.
 74. The peptide combinationof claim 72, wherein the spacer is a dipeptide comprising two acidicamino acids.
 75. The peptide combination of any of claims 63 to 74,wherein the total length of the spacer and the acyl group is about 14 toabout 28 atoms in length.
 76. The peptide combination of any of claims63 to 75, wherein the acyl group is a C12 to C18 fatty acid.
 77. Thepeptide combination of claim 76, wherein the acyl group is C14 or C16.78. The peptide combination of any of claims 1-22, wherein the GIPagonist peptide is an analog of glucagon (SEQ ID NO: 1), with thefollowing modifications: (a) an amino acid modification at position 1that confers GIP agonist activity, (b) a lactam bridge between the sidechains of amino acids at positions i and i+4 or between the side chainsof amino acids at positions j and j+3, wherein i is 12, 13, 16, 17, 20or 24, and wherein j is 17, (c) amino acid modifications at one, two orall of positions 27, 28 and 29, and (d) 1-6 further amino acidmodifications, wherein the EC50 of the analog for GIP receptoractivation is about 10 nM or less.
 79. The peptide combination of claim78 wherein the amino acid modification at position 1 is a substitutionof His with an amino acid lacking an imidazole side chain.
 80. Thepeptide combination of claim 78 or 79 wherein (a) the amino acidmodification at position 1 is a substitution of His with a large,aromatic amino acid, optionally Tyr, (b) the lactam bridge is betweenthe amino acids at positions 16 and 20, wherein one of the amino acidsat positions 16 and 20 is substituted with Glu, and the other of theamino acids at positions 16 and 20 is substituted with Lys, and (c) theMet at position 27 is substituted with a large aliphatic amino acid,optionally Leu, (d) the Asn at position 28 is substituted with a smallaliphatic amino acid, optionally Ala, and (e) the Thr at position 29 issubstituted with a small aliphatic amino acid, optionally Gly.
 81. Thepeptide combination of any of claims 78 to 80 wherein the GIP agonistpeptide comprises one or more of the following modifications: (a) aminoacid modification at position 12, optionally substitution with Ile, (b)amino acid modifications at positions 17 and 18, optionally substitutionwith Q at position 17 and A at position 18, (c) addition of GPSSGAPPPS(SEQ ID NO: 195) to the C-terminus,
 82. The peptide combination of anyof claims 78 to 81 wherein the GIP agonist peptide comprises one or moreof the following modifications: (l) Ser at position 2 substituted withD-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val, or α-amino-N-butyricacid; (m) Tyr at position 10 substituted with Trp, Lys, Orn, Glu, Phe,or Val; (n) Linkage of an acyl group to a Lys at position 10; (o) Lys atposition 12 substituted with Arg; (p) Ser at position 16 substitutedwith Glu, Gln, homoglutamic acid, homocysteic acid, Thr, Gly, or AIB;(q) Arg at position 17 substituted with Gln; (r) Arg at position 18substituted with Ala, Ser, Thr, or Gly; (s) Gln at position 20substituted with Ala, Lys, Citrulline, Arg, Orn, or AIB; (t) Asp atposition 21 substituted with Glu, homoglutamic acid, homocysteic acid;(u) Val at position 23 substituted with Ile; (v) Gln at position 24substituted with Asn, Ala, or AIB; and (w) a conservative substitutionat any of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 24, 27, 28, and 29; or any combination thereof.
 83. The peptidecombination of any of claims 1-22, wherein the GIP agonist peptide is ananalog of glucagon (SEQ ID NO: 1), with the following modifications: (a)an amino acid modification at position 1 that confers GIP agonistactivity, (b) one, two, three, or all of the amino acids at positions16, 20, 21, and 24 of the analog is substituted with anα,α-disubstituted amino acid, (c) amino acid modifications at one, twoor all of positions 27, 28 and 29, and (d) 1-6 further amino acidmodifications, wherein the EC50 of the analog for GIP receptoractivation is about 10 nM or less.
 84. The peptide combination of claim83 wherein the amino acid modification at position 1 is a substitutionof His with an amino acid lacking an imidazole side chain.
 85. Thepeptide combination of claim 83 or 84 wherein (a) the amino acidmodification at position 1 is a substitution of His with a large,aromatic amino acid, optionally Tyr, (b) the α,α-disubstituted aminoacid is AIB, (c) the Met at position 27 is substituted with a largealiphatic amino acid, optionally Leu, (d) the Asn at position 28 issubstituted with a small aliphatic amino acid, optionally Ala, and (e)the Thr at position 29 is substituted with a small aliphatic amino acid,optionally Gly.
 86. The peptide combination of any of claims 83 to 85wherein the GIP agonist peptide comprises one or more of the followingmodifications: (a) amino acid modification at position 12, optionallysubstitution with Ile, (b) amino acid modifications at positions 17 and18, optionally substitution with Q at position 17 and A at position 18,(c) addition of GPSSGAPPPS (SEQ ID NO: 3) to the C-terminus,
 87. Thepeptide combination of any of claims 83 to 86 wherein the GIP agonistpeptide comprises one or more of the following modifications: (a) Ser atposition 2 substituted with D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB,Val, or α-amino-N-butyric acid; (b) Tyr at position 10 substituted withTrp, Lys, Orn, Glu, Phe, or Val; (c) Linkage of an acyl group to a Lysat position 10; (d) Lys at position 12 substituted with Arg; (e) Ser atposition 16 substituted with Glu, Gln, homoglutamic acid, homocysteicacid, Thr, Gly, or AIB; (f) Arg at position 17 substituted with Gln; (g)Arg at position 18 substituted with Ala, Ser, Thr, or Gly; (h) Gln atposition 20 substituted with Ala, Lys, Citrulline, Arg, Orn, or AIB; (i)Asp at position 21 substituted with Glu, homoglutamic acid, homocysteicacid; (j) Val at position 23 substituted with Ile; (k) Gln at position24 substituted with Asn, Ala, or AIB; and (l) a conservativesubstitution at any of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 24, 27, 28, and 29; or any combination thereof. 88.The peptide combination of any of claims 1-22, wherein the GIP agonistpeptide is an analog of glucagon (SEQ ID NO: 1), with the followingmodifications: (a) an amino acid modification at position 1 that confersGIP agonist activity, (b) an amino acid substitution of Ser at position16 with an amino acid of Formula IV:

wherein n is 1 to 7, wherein each of R1 and R2 is independently selectedfrom the group consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group, (c) an aminoacid substitution of the Gln at position 20 with an alpha,alpha-disubstituted amino acid, (d) amino acid modifications at one, twoor all of positions 27, 28 and 29, and (e) 1-6 further amino acidmodifications, wherein the EC50 of the analog for GIP receptoractivation is about 10 nM or less.
 89. The peptide combination of claim88, wherein the amino acid modification at position 1 is a substitutionof His with an amino acid lacking an imidazole side chain.
 90. Thepeptide combination of claim 89, wherein the amino acid lacking animidazole side chain is a large, aromatic amino acid.
 91. The peptidecombination of claim 90, wherein the large, aromatic amino acid is Tyr.92. The peptide combination of any of claims 88 to 91, wherein the aminoacid of Formula IV in (b) is homoLys, Lys, Orn, or 2,4-diaminobutyricacid (Dab).
 93. The peptide combination of any of claims 88 to 92,wherein the alpha, alpha di-substituted amino acid is AIB.
 94. Thepeptide combination of any of claims 88 to 93, wherein (i) the Met atposition 27 is substituted with a large, aliphatic amino acid,optionally Leu, (ii) the Asn at position 28 is substituted with a smallaliphatic amino acid, optionally Ala, or (iii) the Thr at position 29 issubstituted with a small aliphatic amino acid, optionally Gly, orwherein the analog comprises a combination of (i), (ii), and (iii). 95.The peptide combination of any of claims 88 to 94, wherein the GIPagonist peptide comprises the amino acid sequence of GPSSGAPPPS (SEQ IDNO: 3) or XGPSSGAPPPS (SEQ ID NO: 4) C-terminal to the amino acid atposition
 29. 96. The peptide combination of any of claims 88 to 95,wherein the GIP agonist peptide comprises one or more of the followingmodifications: (a) Ser at position 2 substituted with D-Ser, Ala, D-Ala,Gly, N-methyl-Ser, AIB, Val, or α-amino-N-butyric acid; (b) Gln atposition 3 substituted with Glu; (c) substitution of the amino acid Tyrat position 10 with an amino acid comprising a side chain covalentlylinked to an acyl group or alkyl group; (d) addition of an amino acidcomprising a side chain covalently linked to an acyl group or alkylgroup as the C-terminal amino acid of the analog; (e) Lys at position 12substituted with Ile; (f) Arg at position 17 substituted with Gln; (g)Arg at position 18 substituted with Ala; (h) Asp at position 21substituted with Glu; and (i) Gln at position 24 substituted with Asn;97. The peptide combination of claim 95 or 96 wherein the GIP agonistpeptide comprises (a) an amino acid modification at position 2 thatconfers resistance to DPP-IV, and (b) an amino acid at position 40covalently linked to an acyl group or alkyl group.
 98. The peptidecombination of claim 97 comprising a hydrophilic moiety linked to anamino acid at position
 24. 99. The peptide combination of any of claims1-22, wherein the GIP agonist peptide is an analog of glucagon (SEQ IDNO: 1), comprising: (a) an amino acid modification at position 1 thatconfers GIP agonist activity, and (b) an extension of 1 to 21 aminoacids C-terminal to the amino acid at position 29, wherein at least oneof the amino acids of the extension, corresponding to any of positions37-43 relative to SEQ ID NO: 1, is acylated or alkylated, wherein theEC50 of the analog for GIP receptor activation is about 10 nM or less.100. The peptide combination of claim 99, wherein the analog furthercomprises one of the following modifications: (A) the analog comprises alactam bridge between the side chains of amino acids at positions i andi+4 or between the side chains of amino acids at positions j and j+3,wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17; (B) one,two, three, or all of the amino acids at positions 16, 20, 21, and 24 ofthe analog is substituted with an α,α-disubstituted amino acid; or (C)the analog comprises (i) an amino acid substitution of Ser at position16 with an amino acid of Formula IV:

wherein n is 1 to 7, wherein each of R1 and R2 is independently selectedfrom the group consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group; and (ii) anamino acid substitution of the Gln at position 20 with an alpha,alpha-disubstituted amino acid.
 101. The peptide combination of claim 99or 100, comprising further amino acid modifications at one or both ofpositions 27 and
 28. 102. The peptide combination of any of claims 99 to101, wherein the amino acid modification at position 1 is a substitutionof His with an amino acid lacking an imidazole side chain.
 103. Thepeptide combination of claim 102, wherein the amino acid lacking animidazole side chain is a large, aromatic amino acid.
 104. The peptidecombination of claim 103, wherein the large, aromatic amino acid is Tyr.105. The peptide combination of any of claims 99 to 104, wherein the 1to 21 amino acids comprises the amino acid sequence of GPSSGAPPPS (SEQID NO: 3), or XGPSSGAPPPS (SEQ ID NO: 4), wherein X is any amino acid oran amino acid sequence containing one or more conservative substitutionsrelative to SEQ ID NO: 3 or
 4. 106. The peptide combination of claim 76,wherein the 1 to 21 amino acids comprises the amino acid sequence ofGPSSGAPPPS (SEQ ID NO: 3), or XGPSSGAPPPS (SEQ ID NO: 4), wherein X isany amino acid.
 107. The peptide combination of any of claims 99 to 106,wherein the acylated or alkylated amino acid is an amino acid of FormulaI, II, or III.
 108. The peptide combination of claim 107, wherein theacylated or alkylated amino acid is Lys.
 109. The peptide combination ofany of claims 99 to 108, wherein the acylated or alkylated amino acid islocated at any of positions 37, 38, 39, 40, 41, 42 or 43 of the analog.110. The peptide combination of claim 109, wherein the acylated oralkylated amino acid is located at position 40 of the analog.
 111. Thepeptide combination of any of claims 99 to 110, wherein the analogcomprises a lactam bridge between the amino acids at positions 16 and20, wherein one of the amino acids at positions 16 and 20 is substitutedwith Glu, and the other of the amino acids at positions 16 and 20 issubstituted with Lys.
 112. The peptide combination of any of claims 99to 111, wherein the analog comprises a substitution at one, two, threeor all of the amino acids at positions 16, 20, 21 or 24 with an theα,α-disubstituted amino acid is AIB.
 113. The peptide combination ofclaim 112, wherein the analog comprises an AIB at position
 20. 114. Thepeptide combination of any of claims 99 to 113, wherein the analogcomprises a homoLys, Lys, Orn, or 2,4-diaminobutyric acid (Dab) atposition 16 and an AIB at position
 20. 115. The peptide combination ofany of claims 99 to 114, wherein the GIP agonist peptide comprises oneor more of the following modifications: (a) Ser at position 2substituted with D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val, orα-amino-N-butyric acid; (b) Gln at position 3 substituted with Glu; (c)substitution of the amino acid Tyr at position 10 with an amino acidcomprising a side chain covalently linked to an acyl group or alkylgroup; (e) Lys at position 12 substituted with Ile; (f) Arg at position17 substituted with Gln; (g) Arg at position 18 substituted with Ala;(h) Asp at position 21 substituted with Glu; and (i) Gln at position 24substituted with Asn;
 116. The peptide combination of any of claims 99to 115 wherein the GIP agonist peptide comprises an amino acidmodification at position 2 that confers resistance to DPP-IV.
 117. Thepeptide combination of claim 116 wherein the amino acid at position 2 isselected from the group consisting of D-Ser, Ala, D-Ala, Gly,N-methyl-Ser, AIB, Val, or α-amino-N-butyric acid.
 118. The peptidecombination of any of claims 99 to 117, wherein the GIP agonist peptidecomprises up to 6 further amino acid modifications.
 119. The peptidecombination of any of claims 1-22, wherein the GIP agonist peptide is aglucagon analog comprising the amino acid sequence according to any oneof SEQ ID NOS: 327, 328, 329 or 330 and an extension of 1 to 21 aminoacids C-terminal to the amino acid at position 29, wherein the EC50 ofthe analog for GIP receptor activation is about 10 nM or less.
 120. Thepeptide combination of claim 119, wherein the extension of 1 to 21 aminoacids comprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 3) orXGPSSGAPPPS (SEQ ID NO: 4), wherein X is any amino acid, or an aminoacid sequence containing one or more conservative substitutions relativeto SEQ ID NO: 3 or
 4. 121. The peptide combination of claim 119, whereinthe extension of 1 to 21 amino acids comprises the amino acid sequenceof GPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ ID NO: 4), wherein X isany amino acid.
 122. The peptide combination of any of claims 119 to121, wherein at least one of the amino acids of the extension, at aposition corresponding to any of positions 37-43, is acylated oralkylated.
 123. The peptide combination of claim 122, wherein theacylated or alkylated amino acid is located at position 40 of theanalog.
 124. The peptide combination of any of claims 119 to 123,wherein the analog is covalently linked to a hydrophilic moiety at aminoacid position
 24. 125. The peptide combination of claim 124, wherein thehydrophilic moiety is covalently linked to Lys, Cys, Orn, homocysteine,or acetyl-phenylalanine.
 126. The peptide combination of claim 124 or125, wherein the hydrophilic moiety is a polyethylene glycol (PEG). 127.The peptide combination of any of claims 119 to 126, wherein the GIPagonist peptide further comprises up to 6 further amino acidmodifications.
 128. The peptide combination of claim 127 comprising oneor more of the following modifications: (a) the amino acid at position 2is any one of D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val, orα-amino-N-butyric acid; (a) the amino acid at position 10 is Tyr, Trp,Lys, Orn, Glu, Phe, or Val; (b) linkage of an acyl group to a Lys atposition 10; (c) the amino acid at position 12 is Ile, Lys or Arg; (d)the amino acid at position 16 is any one of Ser, Glu, Gln, homoglutamicacid, homocysteic acid, Thr, Gly, or AIB; (e) the amino acid at position17 is Gln or Arg; (f) the amino acid at position 18 is any one of Ala,Arg, Ser, Thr, or Gly; (g) the amino acid at position 20 is any one ofAla, Lys, Citrulline, Arg, Orn, or AIB or another alpha,alpha-disubstituted amino acid; (h) the amino acid at position 21 is anyone of Glu, Asp, homoglutamic acid, homocysteic acid; (i) the amino acidat position 23 is Val or Ile; (j) the amino acid at position 24 is anyone of Gln, Asn, Ala, or AIB; and (k) one or more conservativesubstitutions at any of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 24, 27, 28, and
 29. 129. The peptide combination ofany of the preceding claims, wherein the GIP agonist peptide is: (a) ananalog comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 199-241, 244-264, 266, 292-307, 309-321 and323, (b) an analog comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 267-269, 273-278 and 325; and (c) ananalog comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 105-194.
 130. The peptide combination of claim82 or 87, wherein the acyl group is linked to the Lys via a spacer. 131.The peptide combination of any of claims 96 to 98, 115, and 128, whereinthe acyl group or alkyl group is linked to the amino acid side chain viaa spacer.
 132. The peptide combination of any of claims 99, 122, and123, wherein the acylated or alkylated amino acid at any of positions37-43 relative to SEQ ID NO: 1 is covalently attached to an acyl oralkyl group via a spacer.
 133. The peptide combination of claim 135,wherein the acylated or alkylated amino acid is located at position 40relative to SEQ ID NO:
 1. 134. The peptide combination of claim 129,wherein, when the analog comprises an acyl or alkyl group, the acyl oralkyl group is attached to the analog via a spacer.
 135. The peptidecombination of any of claims 130 to 134, wherein the spacer is 3 to 10atoms in length.
 136. The peptide combination of claim 135, wherein thespacer is an amino acid or dipeptide.
 137. The peptide combination ofclaim 136, wherein the spacer is 6-amino hexanoic acid.
 138. The peptidecombination of claim 136, wherein the spacer is a dipeptide comprisestwo acidic amino acids.
 139. The peptide combination of any of claims130 to 138, wherein the total length of the spacer and the acyl group isabout 14 to about 28 atoms in length.
 140. The peptide combination ofclaims 130 to 139, wherein the acyl group is a C12 to C18 fatty acylgroup.
 141. The peptide combination of claim 140, wherein the acyl groupis a C14 or C16 fatty acyl group.
 142. The peptide combination of any ofthe preceding claims, wherein the EC50 of the GIP agonist peptide forGIP receptor activation is about 1 nM or less.
 143. The peptidecombination of any of the preceding claims, wherein the GIP agonistpeptide has at least about 4% of the activity of wild-type GIP (SEQ IDNO: 2) at the GIP receptor.
 144. The peptide combination of any of thepreceding claims, wherein the EC50 of the GIP agonist peptide for GLP-1receptor activation is about 1 nM or less.
 145. The peptide combinationof any of the preceding claims, wherein the GIP agonist peptide has atleast about 4% of the activity of GLP-1 at the GLP-1 receptor.
 146. Thepeptide combination of any of the preceding claims excluding claim 120,wherein the GIP agonist peptide comprises an amino acid modification atposition 3 and has less than 1% of the activity of glucagon at theglucagon receptor.
 147. The peptide combination of any of the precedingclaims excluding claims 144 and 145, wherein the GIP agonist peptidecomprises an amino acid modification at position 7 and has less thanabout 10% of the activity of GLP-1 at the GLP-1 receptor.
 148. Thepeptide combination of any of the preceding claims, wherein the GIPagonist peptide is covalently linked to a hydrophilic moiety at any ofamino acid positions 19, 20, 23, 24, 27, 32, 43 or the C-terminus. 149.The peptide combination of claim 148, wherein the GIP agonist peptide iscovalently linked to a hydrophilic moiety at amino acid position 27 or43.
 150. The peptide combination of claim 148 or 149, wherein thehydrophilic moiety is covalently linked to Lys, Cys, Orn, homocysteine,or acetyl-phenylalanine.
 151. The peptide combination of any of claims148 to 150, wherein the hydrophilic moiety is a polyethylene glycol(PEG).
 152. The peptide combination of claim 151, wherein the PEG has amolecular weight of about 1,000 Daltons to about 40,000 Daltons. 153.The peptide combination of claim 151, wherein the PEG has a molecularweight of about 20,000 Daltons to about 40,000 Daltons.
 154. The peptidecombination of any of claims 148 to 153 wherein the EC50 of the analogfor GIP receptor activation is about 10 nM or less.
 155. The peptidecombination of any of claims 148 to 153, wherein the analog has at leastabout 0.4% of the activity of wild-type GIP (SEQ ID NO: 2) at the GIPreceptor.
 156. The peptide combination of any of claims 148 to 155,wherein the EC50 of the GIP agonist peptide for GLP-1 receptoractivation is about 10 nM or less.
 157. The peptide combination of anyof claims 148 to 155, wherein the GIP agonist peptide has at least about0.4% of the activity of GLP-1 at the GLP-1 receptor.
 158. The peptidecombination of any of claims 27 to 157, wherein the GIP agonist peptidecomprises the amino acid sequence of any of SEQ ID NOs: 105-194,199-269, 273-278, 292-307, 309-321, 323 and
 325. 159. The peptidecombination of any of claims 1 to 22, wherein the GIP agonist peptidecomprises: (i) SEQ ID NO: 1 with at least one and up to 10 amino acidmodifications, wherein at least one of the amino acid modificationsconfers a stabilized alpha helix structure in the C-terminal portion ofthe GIP agonist peptide; (ii) an extension of 1 to 21 amino acidsC-terminal to the amino acid at position 29, and (iii) at least one ofthe following: (a) at least one of the amino acids of the extensionlocated at any of positions 37-43 (according to the numbering of SEQ IDNO: 1) comprises an acyl or alkyl group which is non-native to anaturally-occurring amino acid, (b) 1-6 amino acids of the extension arepositive-charged amino acids, (c) the GIP agonist peptide comprises anamino acid comprising an acyl or alkyl group, which is non-native to anaturally-occurring amino acid, at position 10 of the GIP agonistpeptide, or (d) a combination of (a), (b), and (c); wherein, when theGIP agonist peptide lacks a hydrophilic moiety, the GIP agonist peptideexhibits at least 0.1% activity of native GIP at the GIP receptor. 160.The peptide combination of claim 159, wherein (i) the GIP agonistpeptide comprises an intramolecular bridge between the side chains of anamino acid at position i and an amino acid at position i+4 or betweenthe side chains of amino acids at positions j and j+3, wherein i is 12,13, 16, 17, 20 or 24 and j is 17, (ii) one, two, three or more ofpositions 16, 20, 21 or 24 of the GIP agonist peptide are substitutedwith an α,α-disubstituted amino acid, or (iii) both (i) and (ii). 161.The peptide combination of claim 159 or 160, wherein the GIP agonistpeptide comprises Glu at position 16 and Lys at position 20, whereinoptionally a lactam bridge links the Glu and the Lys.
 162. The peptidecombination of claim 161, wherein the GIP agonist peptide comprises oneor more of: Gln at position 17, Ala at position 18, Glu at position 21,Ile at position 23, and Ala or Cys at position 24, or one or moreconservative amino acid substitutions thereof.
 163. The peptidecombination of any of claims 159 to 162, wherein the GIP agonist peptidecomprises a C-terminal amide.
 164. The peptide combination of any ofclaims 159 to 163 wherein the GIP agonist peptide comprises an aminoacid substitution at position 1, position 2, or positions 1 and 2,wherein the amino acid substitution(s) achieve DPP-IV proteaseresistance.
 165. The peptide combination of claim 164, wherein the Hisat position 1 of the GIP agonist peptide is substituted with an aminoacid selected from the group consisting of: D-histidine, alpha,alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine,alpha-methyl histidine, imidazole acetic acid, desaminohistidine,hydroxyl-histidine, acetyl-histidine and homo-histidine.
 166. Thepeptide combination of claim 164 or 165, wherein the Ser at position 2is substituted with an amino acid selected from the group consisting of:D-serine, alanine, D-alanine, valine, glycine, N-methyl serine, N-methylalanine, and amino isobutyric acid (AIB).
 167. The peptide combinationof any of claims 159 to 164, wherein the amino acid at position 1 of theanalog is not a large, aromatic amino acid.
 168. The peptide combinationof claim 167, wherein the amino acid at position 1 of the analog is notTyr.
 169. The peptide combination of any of claims 159 to 168, whereinthe GIP agonist peptide comprises one or more of the followingmodifications: a. Ser at position 2 substituted with Ala; b. Gln atposition 3 substituted with Glu or a glutamine analog; c. Thr atposition 7 substituted with a Ile; d. Tyr at position 10 substitutedwith Trp or an amino acid comprising an acyl or alkyl group which isnon-native to a naturally-occurring amino acid; e. Lys at position 12substituted with Ile; f. Asp at position 15 substituted with Glu; g. Serat position 16 substituted with Glu; h. Gln at position 20 substitutedwith Ser, Thr, Ala, AIB; i. Gln at position 24 substituted with Ser,Thr, Ala, AIB; j. Met at position 27 substituted with Leu or Nle; k. Asnat position 29 substituted with a charged amino acid, optionally, Asp orGlu; and l. Thr at position 29 substituted with Gly or a charged aminoacid, optionally, Asp or Glu.
 170. The peptide combination of any ofclaims 159 to 169, wherein the GIP agonist peptide comprises (i) theamino acid sequence of GPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ IDNO: 4), wherein X is any amino acid, (ii) an amino acid sequence whichhas at least 80% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, or(iii) the amino acid sequence of (i) or (ii) with one or moreconservative amino acid substitutions, wherein the amino acid sequenceis C-terminal to the amino acid at position 29 of the GIP agonistpeptide.
 171. The peptide combination of claim 170, comprising the aminoacid sequence of GPSSGAPPPS (SEQ ID NO: 3) or XGPSSGAPPPS (SEQ ID NO:4), wherein X is any amino acid, C-terminal to the amino acid atposition 29 of the GIP agonist peptide.
 172. The peptide combination ofany of claims 159 to 171, wherein the amino acid comprising an acyl oralkyl group is an amino acid of Formula I, II, or III.
 173. The peptidecombination of claim 172, wherein the amino acid comprising an acyl oralkyl group is Lys.
 174. The peptide combination of any of claims 159 to173, wherein the amino acid comprising an acyl or alkyl group is locatedat any of positions 37, 38, 39, 40, 41, 42 or 43 of the GIP agonistpeptide.
 175. The peptide combination of claim 174, wherein the aminoacid comprising an acyl or alkyl group is located at position 40 of theGIP agonist peptide.
 176. The peptide combination of any of claims 159to 175, wherein the acyl group is a C4 to C30 fatty acyl group.
 177. Thepeptide combination of any of claims 159 to 176, wherein the acyl oralkyl group is covalently attached to the side chain of the amino acidvia a spacer.
 178. The peptide combination of claim 177, wherein thespacer is 3 to 10 atoms in length.
 179. The peptide combination of claim178, wherein the spacer is an amino acid or dipeptide.
 180. The peptidecombination of claim 179, wherein the spacer is 6-amino hexanoic acid.181. The peptide combination of claim 179, wherein the spacer is adipeptide comprising two acidic amino acids.
 182. The peptidecombination of any of claims 177 to 181, wherein the total length of thespacer and the acyl group is about 14 to about 28 atoms in length. 183.The peptide combination of any of claims 159 to 182, wherein the acylgroup is a C12 to C18 fatty acyl group.
 184. The peptide combination ofclaim 183, wherein the acyl group is a C14 or C16 fatty acyl group. 185.The peptide combination of any of claims 159 to 184, wherein the 1-6positive-charged amino acids are located at any of located at any ofpositions 37, 38, 39, 40, 41, 42, and 43 (according to the numbering ofSEQ ID NO: 1) of the GIP agonist peptide.
 186. The peptide combinationof claim 185, comprising a positive-charged amino acid at position 40.187. The peptide combination of any of claims 159 to 186, wherein thepositive-charged amino acid comprises the structure of Formula IV:

wherein n is 1 to 16, or 1 to 10, or 1 to 7, or 1 to 6, or 2 to 6, or 2or 3 or 4 or 5, each of R₁ and R₂ is independently selected from thegroup consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group.
 188. Thepeptide combination of claim 187, wherein the amino acid of Formula IVis Orn, Dab, Lys, D-Lys, or homoLys.
 189. The peptide combination of anyof claims 27-188, wherein the GIP agonist peptide is covalently linkedto a hydrophilic moiety at any of amino acid positions 19, 20, 23, 24,27, 32, 43 or the C-terminus.
 190. The peptide combination of claim 189,wherein the GIP agonist peptide is covalently linked to a hydrophilicmoiety at amino acid position 24 or
 40. 191. The peptide combination ofclaim 189 or 190, wherein the hydrophilic moiety is covalently linked toLys, Cys, Orn, homocysteine, or acetyl-phenylalanine.
 192. The peptidecombination of any of claims 189 to 191, wherein the hydrophilic moietyis a polyethylene glycol (PEG).
 193. The peptide combination of claim192, wherein the PEG has a molecular weight of about 1,000 Daltons toabout 40,000 Daltons.
 194. The peptide combination of claim 192, whereinthe PEG has a molecular weight of about 20,000 Daltons to about 40,000Daltons.
 195. The peptide combination of any of claims 27-194, whereinthe amino acid at position 1 is deleted or substituted with a small,aliphatic amino acid.
 196. The peptide combination of claim 195, whereinthe small aliphatic amino acid is Ala.
 197. The peptide combination ofany of claims 159 to 196, wherein the GIP agonist peptide comprises theamino acid sequence of any of SEQ ID NOs: 1057-1069.
 198. The peptidecombination of any of the preceding claims, wherein the glucagonantagonist peptide comprises the sequence of SEQ ID NO: 1142, or an oxyderivative thereof.
 199. The peptide combination of claim 198,comprising the sequence of SEQ ID NO:
 1142. 200. The peptide combinationof claim 198 or 199, wherein the C-terminal amino acid of the glucagonantagonist peptide has an amide group in place of the carboxylic acidgroup that is present on the native amino acid.
 201. The peptidecombination of claim 198 or 199, wherein the amino acid at position 4 isaspartic acid.
 202. The peptide combination of claim 198 or 199, whereinthe glucagon antagonist peptide comprises the amino acid of SEQ ID NO:1119 fused to the carboxy terminal amino acid of SEQ ID NO:
 1142. 203.The peptide combination of claim 198 or 199, wherein the glucagonantagonist peptide comprises a hydrophilic moiety covalently bound to anamino acid residue at position 11, 16 or 19 of SEQ ID NO: 1142, or atthe N- or C-terminal amino acid of the glucagon antagonist peptide, andpharmaceutically acceptable salts of said glucagon peptide.
 204. Thepeptide combination of claim 203, wherein said hydrophilic moiety is aplasma protein or the Fc portion of an immunoglobin.
 205. The peptidecombination of claim 203, wherein the hydrophilic moiety is polyethyleneglycol.
 206. The peptide combination of claim 205, wherein thepolyethylene glycol chain has a molecular weight of at least about20,000 Daltons.
 207. The peptide combination of claim 205, wherein thepolyethylene glycol chain has a molecular weight selected from the rangeof about 1,000 to about 5,000 Daltons.
 208. The peptide combination ofclaim 205, wherein the antagonist comprises the sequence of SEQ ID NO:1109, SEQ ID NO: 1110, SEQ ID NO: 1111, SEQ ID NO: 1112, SEQ ID NO:1116, SEQ ID NO: 1117, SEQ ID NO: 1118, SEQ ID NO: 1143, SEQ ID NO: 1144or SEQ ID NO:
 1145. 209. The peptide combination of claim 198 or 199,wherein the glucagon antagonist peptide comprises the sequence of SEQ IDNO: 1146 or SEQ ID NO:11
 47. 210. The peptide combination of claim 198or 199, wherein (a) when the amino acid at position 23 is Asn, the aminoacid at position 24 is selected from the group consisting of asparticacid or glutamic acid, and (b) when the amino acid at position 24 isThr, the amino acid at position 23 is selected from the group consistingof aspartic acid or glutamic acid.
 211. The peptide combination of claim198 or 199, wherein the glucagon antagonist peptide further comprisesone to two amino acids added to the carboxy terminus of the glucagonantagonist peptide of SEQ ID NO: 1142, wherein said amino acids added tothe carboxy terminus are independently selected from the groupconsisting of aspartic acid or glutamic acid.
 212. The peptidecombination of claim 198 or 199, wherein the amino acid at position 10of SEQ ID NO: 1142 is selected from the group consisting of Glu, cysteicacid, homoglutamic acid or homocysteic acid.
 213. The peptidecombination of claim 198 or 199, wherein the glucagon antagonist peptidecomprises the sequence of SEQ ID NO: 1107, SEQ ID NO: 1108, SEQ ID NO:1136, SEQ ID NO: 1137, SEQ ID NO: 1138, SEQ ID NO: 1139, SEQ ID NO: 1140and SEQ ID NO:
 1141. 214. The peptide combination of claim 213, whereinthe glucagon antagonist peptide comprises the amino acid of SEQ ID NO:1119 or SEQ ID NO: 1153 fused to the carboxy terminal amino acid of saidglucagon antagonist.
 215. The peptide combination of claim 213, whereinthe glucagon antagonist peptide comprises a hydrophilic moietycovalently bound to an amino acid residue at position 11, 16 or 19 ofSEQ ID NO: 1142, or to the N- or C-terminal amino acid of the glucagonantagonist and pharmaceutically acceptable salts of said glucagonpeptide.
 216. The peptide combination of claim 215, wherein thehydrophilic moiety is polyethylene glycol.
 217. The peptide combinationof claim 216 wherein the glucagon antagonist peptide has the sequence ofSEQ ID NO: 1141, wherein the amino acid at positions 4 is aspartic acidand position 10 is glutamic acid.
 218. The peptide combination of claim213, wherein the glucagon antagonist peptide comprises the sequence ofSEQ ID NO: 1108, SEQ ID NO: 1137 or SEQ ID NO:
 1138. 219. The peptidecombination of claim 218, wherein the glucagon antagonist peptidefurther comprises the amino acid of SEQ ID NO: 1119 fused to the carboxyterminal amino acid of said glucagon antagonist.
 220. The peptidecombination of claim 218, wherein the glucagon antagonist peptidefurther comprises a hydrophilic moiety covalently bound to an amino acidresidue at position 11, 16 or 19, and pharmaceutically acceptable saltsof said glucagon peptide.
 221. The peptide combination of claim 198 or199, wherein the peptide comprises a derivative of the peptide of SEQ IDNO: 1142, wherein the glucagon peptide differs from the peptide of SEQID NO: 1142 by amino acid substitutions at one to three amino acidpositions selected from positions 5, 6, 8, 9, 12, 13 and
 14. 222. Thepeptide combination of any of the preceding claims, wherein the oxyderivative is an ester or ether of SEQ ID NO:
 1142. 223. The peptidecombination of any of claims 1 to 197, wherein the glucagon antagonistpeptide comprises the amino acid sequence of native glucagon modified bydeletion of two to five amino acid residues from the N-terminus of SEQID NO: 1, and substitution of the aspartic acid residue at position nineof SEQ ID NO: 1 with glutamic acid, homoglutamic acid, β-homoglutamicacid, a sulfonic acid derivative of cysteine, or an alkylcarboxylatederivative of cysteine having the structure of:

wherein X₅ is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl.
 224. Thepeptide combination of claim 223, wherein the sulfonic acid derivativeof cysteine is homocysteic acid.
 225. The peptide combination of claim223, wherein X₅ is C_(i) alkyl or C₂ alkyl.
 226. The peptide combinationof any of claims 223 to 225, wherein the amino acid sequence of nativeglucagon is further modified by up to three amino acid modifications.227. The peptide combination of claim 226, wherein the up to three aminoacid modifications are selected from the group consisting of: A.substitution of one or two amino acids at positions 10, 20, and 24,(according to the amino acid numbering of SEQ ID NO: 1), or the N- orC-terminal amino acid of the glucagon antagonist peptide with an aminoacid comprising an acyl or alkyl group; B. substitution of one or twoamino acids at positions 16, 17, 20, 21, and 24 (according to the aminoacid numbering of SEQ ID NO: 1), or the N- or C-terminal amino acid ofthe glucagon antagonist peptide with an amino acid selected from thegroup consisting of: Cys, Lys, ornithine, homocysteine, andacetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group iscovalently bonded to a hydrophilic moiety; C. addition of an amino acidcovalently bonded to a hydrophilic moiety to the N- or C-terminus of theglucagon antagonist peptide; D. substitution of Asp at position 15(according to the numbering of SEQ ID NO: 1) with cysteic acid, glutamicacid, homoglutamic acid, and homocysteic acid; E. substitution of Ser atposition 16 (according to the numbering of SEQ ID NO: 1) with cysteicacid, glutamic acid, homoglutamic acid, and homocysteic acid; F.substitution with AIB at one or more of positions 16, 20, 21, and 24according to the amino acid numbering of SEQ ID NO: 1; G. deletion ofthe amino acid at position 29 or the amino acids at positions 28 and 29,according to the numbering of SEQ ID NO: 1; H. substitution of each orboth of the Asn at position 28 and the Thr at position 29 (according tothe amino acid numbering of SEQ ID NO: 1) with charged amino acids;and/or addition of one to two charged amino acids at the C-terminus ofSEQ ID NO: 1; I. substitution of the Met at position 27 (according tothe numbering of SEQ ID NO: 1) with Leu or norleucine; J. addition of apeptide having the amino acid sequence of any of SEQ ID NOs: 1119-1121and 1153 to the C-terminus of SEQ ID NO: 1; wherein Thr at position 29(according to the numbering of SEQ ID NO: 1) is Thr or Gly; and K.replacement of the C-terminal carboxylate with an amide or ester. 228.The peptide combination of claim 227, wherein the glucagons antagonistpeptide comprises an amino acid modification of A, B, or C, as describedin claim 227, or a combination thereof.
 229. The peptide combination ofclaim 228, further comprising an amino acid modification of any of D toK, as described in claim 227, or a combination thereof.
 230. The peptidecombination of any of the claims 1-197, wherein the glucagon antagonistpeptide comprises the general structure of A-B-C, wherein A is selectedfrom the group consisting of: (i) phenyl lactic acid (PLA); (ii) an oxyderivative of PLA; (iii) a peptide of 2 to 6 amino acids in which twoconsecutive amino acids of the peptide are linked via an ester or etherbond; B represents amino acids i to 26 of SEQ ID NO: 1, wherein i is 3,4, 5, 6, or 7, optionally comprising one or more amino acidmodifications selected from the group consisting of: (iv) Asp atposition 9 (according to the amino acid numbering of SEQ ID NO: 1) issubstituted with a Glu, a sulfonic acid derivative of Cys, homoglutamicacid, β-homoglutamic acid, or an alkylcarboxylate derivative of cysteinehaving the structure of:

wherein X₅ is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. (v)substitution of one or two amino acids at positions 10, 20, and 24,(according to the amino acid numbering of SEQ ID NO: 1) with an aminoacid covalently attached to an acyl or alkyl group via an ester, ether,thioether, amide, or alkyl amine linkage; (vi) substitution of one ortwo amino acids at positions 16, 17, 20, 21, and 24 (according to theamino acid numbering of SEQ ID NO: 1) with an amino acid selected fromthe group consisting of: Cys, Lys, ornithine, homocysteine, andacetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group iscovalently attached to a hydrophilic moiety; (vii) Asp at position 15(according to the numbering of SEQ ID NO: 1) is substituted with cysteicacid, glutamic acid, homoglutamic acid, and homocysteic acid; (viii) Serat position 16 (according to the numbering of SEQ ID NO: 1) issubstituted with cysteic acid, glutamic acid, homoglutamic acid, andhomocysteic acid; (ix) substitution with AIB at one or more of positions16, 20, 21, and 24 according to the amino acid numbering of SEQ ID NO:1; and C is selected from the group consisting of: (x) X; (xi) X-Y;(xii) X-Y-Z; and (xiii) X-Y-Z-R10, wherein X is Met, Leu, or Nle; Y isAsn or a charged amino acid; Z is Thr, Gly, Cys, Lys, ornithine (Orn),homocysteine, acetyl phenylalanine (Ac-Phe), or a charged amino acid;wherein R10 is selected from a group consisting of SEQ ID NOs: 1119-1121and 1153; and (xiv) any of (x) to (xiii) in which the C-terminalcarboxylate is replaced with an amide or ester.
 231. The peptidecombination of claim 230, comprising an intramolecular bridge, or analpha, alpha di-substituted amino acid, or an acidic amino acid atposition 16 (according to the numbering of SEQ ID NO: 1).
 232. Thepeptide combination of claim 230 or 231, comprising Arg at position 17is replaced with Gln, Arg at position 18 is replaced with Ala, Asp atposition 21 is replaced with Glu, Val at position 23 is replaced withIle, and Gln at position 24 is replaced with Ala (according to aminoacid numbering of SEQ ID NO: 1).
 233. The peptide combination of any ofclaims 230 to 232, wherein Ser at position 16 is replaced with Glu, Glnat position 20 is replaced with Glu, or Gln at position 24 is replacedwith Glu (according to the amino acid numbering of SEQ ID NO: 1). 234.The peptide combination of any of claims 198 to 233, wherein theglucagon antagonist peptide comprises the amino acid sequence of any ofSEQ ID NOs: 1162, 1164-1167 and 1171 or as described in any of Tables Dto L.
 235. The peptide combination of any of claims 1 to 197, whereinthe glucagon antagonist peptide is a glucagon antagonist/GLP-1 agonistcomprising the sequence of SEQ ID NO: 1251, wherein the amino acids atpositions 4 and 7, positions 7 and 11, positions 11 and 15, positions 15and 19, or positions 19 and 23 of SEQ ID NO: 1251 are linked via alactam bridge, or an oxy derivative of the glucagon antagonist/GLP-1agonist.
 236. The peptide combination of claim 240, wherein the glucagonantagonist/GLP-1 agonist further comprises the sequence of SEQ ID NO:1250 bound to amino acid 24 of said glucagon antagonist/GLP-1 agonist.237. The peptide combination of claim 235 or 236, wherein the glucagonantagonist/GLP-1 agonist further comprises a hydrophilic moietycovalently bound to an amino acid residue at position 16 or 19, andpharmaceutically acceptable salts of said glucagon antagonist/GLP-1agonist.
 238. The peptide combination of claim 237, wherein saidhydrophilic moiety is polyethylene glycol.
 239. The peptide combinationof claim 235 wherein the glucagon antagonist/GLP-1 agonist comprises asequence selected from the group consisting of SEQ ID NO: 1212, SEQ IDNO: 1213 and SEQ ID NO:
 1214. 240. The peptide combination of claim 238wherein the polyethylene glycol chain has a molecular weight selectedfrom the range of about 1,000 to about 5,000 Daltons.
 241. The peptidecombination of claim 238 wherein the polyethylene glycol chain has amolecular weight of at least about 20,000 Daltons.
 242. The peptidecombination of claim 235, wherein said hydrophilic moiety is a plasmaprotein or the Fc portion of an immunoglobin.
 243. The peptidecombination of claim 235 further comprising the sequence of SEQ ID NO:1221 bound to amino acid 24 of said glucagon antagonist/GLP-1 agonist.244. The peptide combination of claim 238 wherein the amino acid atposition 4 is aspartic acid, the amino acid at position 22 is methionineand the amino acid at position 10 is aspartic acid.
 245. The peptidecombination of claim 235 wherein the C-terminal amino acid of theglucagon antagonist/GLP-1 agonist comprises an amide group in place ofthe carboxylic acid group of the native amino acid.
 246. The peptidecombination of claim 235 wherein the glucagon antagonist/GLP-1 agonistcomprises a sequence selected from the group consisting of SEQ ID NO:1205, SEQ ID NO: 1206, SEQ ID NO: 1207, SEQ ID NO: 1208, SEQ ID NO:1209, SEQ ID NO: 1222, SEQ ID NO: 1223, SEQ ID NO: 1224 and SEQ ID NO:1225.
 247. The peptide combination of claim 246 wherein the glucagonantagonist/GLP-1 agonist comprises a sequence selected from the groupconsisting of SEQ ID NO: 1222, SEQ ID NO: 1223, SEQ ID NO: 1224 and SEQID NO: 1225, wherein the amino acids at position 4 and 10 are bothaspartic acid.
 248. The peptide combination of claim 246 wherein theglucagon antagonist/GLP-1 agonist comprises a sequence selected from thegroup consisting of SEQ ID NO: 1216, SEQ ID NO: 1217, SEQ ID NO: 1218and SEQ ID NO:
 1219. 249. The peptide combination of claim 247 whereinan amino acid at position 16 or 19 further comprises a polyethyleneglycol chain covalently bound to the amino acid residue.
 250. Thepeptide combination of claim 249 wherein the glucagon antagonist/GLP-1agonist further comprises the sequence of SEQ ID NO: 1221 bound to aminoacid 24 of said glucagon antagonist/GLP-1 agonist.
 251. The peptidecombination of claim 254 or 255 wherein the polyethylene glycol chainhas a molecular weight selected from the range of about 1,000 to about5,000 Daltons.
 252. The peptide combination of claim 254 or 255 whereinthe polyethylene glycol chain has a molecular weight of at least about20,000 Daltons.
 253. The peptide combination of claim 247 wherein theglucagon antagonist/GLP-1 agonist comprises a sequence selected from thegroup consisting of SEQ ID NO: 1245 and SEQ ID NO:
 1246. 254. Thepeptide combination of claims 1 to 253, wherein the amino acids atpositions 7 and 11 or 11 and 15 of SEQ ID NO: 1251 are linked via alactam bridge.
 255. The peptide combination of any of claims 1-197,wherein the glucagon antagonist peptide is a glucagon antagonist/GLP-1agonist peptide, said peptide exhibiting at least 90% of the maximumagonism of native GLP-1 at the GLP-1 receptor, and exhibiting glucagonantagonist activity, that reduces the maximum glucagon-induced cAMPproduction by the glucagon receptor by at least about 80%, as measuredby cAMP production in an in vitro assay.
 256. The peptide combination ofclaim 255 wherein the peptide comprises a derivative of the peptide ofSEQ ID NO: 1251, wherein the glucagon peptide differs from the peptideof SEQ ID NO: 1251 by amino acid substitutions at one to three aminoacid positions selected from positions 1, 5, 6, 8, 9, 12, 13 and 14.257. The peptide combination of claim 263 wherein the substitutions areconservative amino acid substitutions.
 258. The peptide combination ofclaim 263 wherein the peptide comprises the sequence of SEQ ID NO: 1247or SEQ ID NO:
 1248. 259. The peptide combination of claim 258 whereinthe amino acid at position 4 is glutamic acid.
 260. The peptidecombination of any of claims 1 to 259 comprising (1) an intramolecularbridge, or an alpha, alpha-di-substituted amino acid, or an acidic aminoacid at position 16 (according to the numbering of SEQ ID NO: 1), or acombination thereof, (2) a C-terminal amide or ester in place of aC-terminal carboxylate, and (3) a general structure of A-B-C, wherein Ais selected from the group consisting of (i) PLA; (ii) an oxy derivativeof PLA; and (iii) a peptide of 2 to 6 amino acids in which twoconsecutive amino acids of the peptide are linked via an ester or etherbond; wherein B represents amino acids p to 26 of SEQ ID NO: 1, whereinp is 3, 4, 5, 6, or 7, optionally comprising one or more amino acidmodifications selected from the group consisting of: (iv) Asp atposition 9 (according to the amino acid numbering of SEQ ID NO: 1) issubstituted with a Glu, a sulfonic acid derivative of Cys, homoglutamicacid, β-homoglutamic acid, or an alkylcarboxylate derivative of cysteinehaving the structure of:

wherein X₅ is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl; (v)substitution of one or two amino acids at positions 10, 20, and 24,(according to the amino acid numbering of SEQ ID NO: 1) with an aminoacid covalently attached to an acyl or alkyl group via an ester, ether,thioether, amide, or alkyl amine linkage; (vi) substitution of one ortwo amino acids at positions 16, 17, 20, 21, and 24 (according to theamino acid numbering of SEQ ID NO: 1) with an amino acid selected fromthe group consisting of: Cys, Lys, ornithine, homocysteine, andacetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group iscovalently attached to a hydrophilic moiety; (vii) Asp at position 15(according to the numbering of SEQ ID NO: 1) is substituted with cysteicacid, glutamic acid, homoglutamic acid, and homocysteic acid; (viii) Serat position 16 (according to the numbering of SEQ ID NO: 1) issubstituted with cysteic acid, glutamic acid, homoglutamic acid, andhomocysteic acid; (ix) Arg at position 17 is replaced with Gln, Arg atposition 18 is replaced with Ala, Asp at position 21 is replaced withGlu, Val at position 23 is replaced with Ile, and Gln at position 24 isreplaced with Ala (according to amino acid numbering of SEQ ID NO: 1);(x) Ser at position 16 is replaced with Glu, Gln at position 20 isreplaced with Glu, or Gln at position 24 is replaced with Glu (accordingto the amino acid numbering of SEQ ID NO: 1); wherein C is selected fromthe group consisting of: (vii) X; (viii) X-Y; (ix) X-Y-Z; (x) X-Y-Z-R10;wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid; Z isThr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine(Ac-Phe), or a charged amino acid; wherein R10 is selected from a groupconsisting of SEQ ID NOs: 1221, 1226, 1227, and
 1250. 261. The peptidecombination of claim 260, wherein the acidic amino acid is an amino acidcomprising a side chain sulfonic acid or a side chain carboxylic acid.262. The peptide combination of claim 261, wherein the acidic amino acidis selected from the group consisting of Glu, Asp, homoglutamic acid, asulfonic acid derivative of Cys, cysteic acid, homocysteic acid, Asp, oran alkylated derivative of Cys having the structure of

wherein X₅ is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl.
 263. Thepeptide combination of any of claims 260 to 262, wherein the oxyderivative of PLA is an ester of PLA or ether of PLA.
 264. The peptidecombination of any of claims 260 to 263, wherein the oxy derivative ofPLA is PLA linked to an amino acid, peptide, hydrophilic polymer, acylgroup, or alkyl group via an ester bond or ether bond.
 265. The peptidecombination of claim 264, wherein the oxy derivative of PLA is adepsipeptide comprising PLA covalently linked via an ester bond to anamino acid or a peptide.
 266. The peptide combination of claim 265,wherein the amino acid is Xaa3, or wherein the peptide comprisesXaa2-Xaa3 or Xaa1-Xaa2-Xaa3, wherein Xaa3 is Gln or Glu, Xaa2 isselected from a group consisting of: Ser, D-serine, D-alanine, valine,glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid(AIB); and Xaa₁ is selected from a group consisting of: His,D-histidine, alpha, alpha-dimethyl imidiazole acetic acid (DMIA),N-methyl histidine, alpha-methyl histidine, imidazole acetic acid,desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine.
 267. The peptide combination of any of claims 260 to262, wherein the peptide of (iii) comprises amino acids q to 6 of SEQ IDNO: 1, wherein q is 1, 2, 3, 4, or
 5. 268. The peptide combination ofany of claims 260 to 267, wherein B represents amino acids 7 to 26 ofSEQ ID NO:
 1. 269. The peptide combination of any of claims 260 to 268,wherein B comprises the amino acid modification designated as (v) or(vi), or a combination thereof.
 270. The peptide combination of claim269, wherein B further comprises one or more amino acid modificationsselected from the group consisting of (iv), (vii), (viii), (ix), (x),and a combination thereof.
 271. The peptide combination of any of claims260 to 270, wherein, when Y or Z is a charged amino acid, the chargedamino acid is selected from a group consisting of: Lys, Arg, His, Asp,and Glu.
 272. The peptide combination of any of claims 260 to 271,wherein the glucagon antagonist comprises one to two charged amino acidsC-terminal to Z, when C comprises X-Y-Z.
 273. The peptide combination ofany of claims 260 to 272, wherein the glucagon antagonist comprises ahydrophilic moiety covalently bound to an amino acid residue at position16, 21, or 24 according to the amino acid numbering of SEQ ID NO: 1, orthe C-terminal residue of the peptide.
 274. The peptide combination ofany of claims 260 to 273, wherein the glucagon antagonist comprises anamino acid covalently attached to an acyl group or alkyl group via anester, ether, thioether, amide, or alkyl amine linkage, wherein theamino acid is at position 10, 20, or 24 (according to the amino acidnumbering of SEQ ID NO: 1), or is the N- or C-terminal residue of thepeptide.
 275. The peptide combination of any of claims 260 to 274,wherein the intramolecular bridge is a lactam bridge.
 276. The peptidecombination of claim 275, wherein the glucagon antagonist comprises alactam bridge between the amino acids at positions 9 and 12, the aminoacids at positions 12 and 16, the amino acids at positions 16 and 20,the amino acids at positions 20 and 24, or the amino acids at positions24 and 28 (according to the amino acid numbering of SEQ ID NO: 1). 277.The peptide combination of claim 276, wherein the glucagon antagonistcomprises a lactam bridge between the amino acids at positions 12 and 16or at positions 16 and
 20. 278. The peptide combination of any of claims260 to 277, wherein the glucagon antagonist comprises AIB at position16, 20, 21, or 24 (according to the amino acid numbering of SEQ ID NO:1).
 279. The peptide combination of any of claims 260 to 278, whereinthe glucagon antagonist comprises the amino acid sequence of any of SEQID NOs: 1260-1270, 1273-1278, 1280-1288, 1290-1296, 1303, 1304, 1306,and 1314-1318, or comprising the amino acid sequence of any of Peptides2-6 of Table A, Peptides 1-8 of Table B, and Peptides 2-6, 8, and 9 ofTable C.
 280. The peptide combination of any of the proceeding claims,wherein: (i) wherein the GIP agonist peptide comprises the amino acidsequence of any of SEQ ID NOs: 105-194, 199-269, 273-278, 292-307,309-321, 323 and 325 or any of SEQ ID NOs: 1057-1069; (ii) wherein theglucagon antagonist peptide comprises the amino acid sequence of any ofSEQ ID NOs: 1162, 1164-1167 and 1171 or as described in any of Tables Dto L or the amino acid sequence of any of SEQ ID NOs: 1260-1270,1273-1278, 1280-1288, 1290-1296, 1303, 1304, 1306, and 1314-1318, orcomprising the amino acid sequence of any of Peptides 2-6 of Table A,Peptides 1-8 of Table B, and Peptides 2-6, 8, and 9 of Table C; or (iii)both (i) and (ii).
 281. The peptide combination of any of claims 8-280,wherein the conjugate is in admixture with a pharmaceutically acceptablecarrier to form a pharmaceutical composition.
 282. A method of treatingdiabetes in a patient, comprising administering to the patient aneffective amount of the pharmaceutical composition of any of claim 3 or281.
 283. A method of causing temporary paralysis of the intestinaltract in a patient, comprising administering to the patient an effectiveamount of the pharmaceutical composition of claim 3 or
 281. 284. Amethod of reducing weight gain or inducing weight loss in a patient,comprising administering to the patient an effective amount of thepharmaceutical composition of claim 3 or
 281. 285. A glucagon analogpeptide comprising the amino acid sequence of any of SEQ ID NOs: 10-36.286. Use of the glucagon analog peptide of claim 286 in the manufactureof a medicament for treating diabetes in a patient, causing temporaryparalysis of the intestinal tract in a patient, or reducing weight gainor inducing weight loss in a patient.